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Antiulcer activity of the calcium antagonist propyl-mcthylcnedioxyindene

Wong, Wai-shiu Fred, Ph.D.

The Ohio State University, 1990

UMI 300 N. ZccbRd. Ann Arbor, MI 48106

ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST

FROPYL-METHYLENEDIOXYINDENE

DISSERTATION

Presented In Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Wai-shiu Fred Wong, U.S. P h arm ., M.S.

t\t ijt >Jt >{r

The Ohio State University

1990

Dissertation Committee: Approved By

Dr. Ralf G. Rahwan

Dr. Robert L. Stephens Jr.

Dr. Norman J. Uretsky A dvisor Dr. Lane J. Wallace College of Pharmacy To God for giving me wisdom to explore the physical nature of man whom He creates,

and

to my parents for their sacrifices, and their unceasing love, support and encouragement,

and

to Amanda for her love, concern and understanding.

11 ACKNOWLEDGMENTS

I want to express my sincere appreciation and gratitude to:

Dr. Ralf G. Rahwan for his guidance and encouragement, and his persistent enthusiasm and confidence in my research works.

Drs. Robert L. Stephens, Jr., Norman J. Uretsky, and Lane J. Wallace for their helpful suggestions and constructive criticisms, and special thanks to Dr. Wallace for his excellent advice in statistics and computer instructions, and to Dr. Stephens for his active participation in my experiments.

Dr. McKay for his guidance in taking photomicrographs from microscopic s lid e s .

Drs. Dennis R. Feller, Popat N. Patil and Allan M. Burkman for their constant concerns and suggestions.

iii VITA

November 16, 1961 ...... Born - Kowloon, Hong Kong

1985 ...... B.S, in Pharmacy, The St. John's University, Jamaica, New York

1985 - 1990 ...... Graduate Teaching Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio

1988 ...... M.S . in Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio

PUBLICATIONS

Wong, W.S.F.: Intracellular actions of calcium antagonists in saponin­ skinned vascular smooth muscle. M.Sc. Thesis, The Ohio State University, Columbus, Ohio, 1988.

Wong, W.S.F. and Rahwan, R.G.: Pharamcolglcal actions of the calcium antagonist propyl-methylenedioxylndene in skinned vascular smooth muscle. Can. J, Physiol. Pharmacol. 66: 1041-1047, 1988.

Rahw an, R.G. and Wong, W.S.F. : Site of action of the calcium antagonist propyl-methylenedioxylndene (pr-MDI) in skinned vascular smooth muscle. 4th Int. Congr. Cell Biol. Abst. P10.1.2, 1988.

Wong, W.S.F. and Rahwan, R.G.: Examination of the potential antiepileptic activity of calcium antagonists with different sites of action. Gen. Pharmacol. 20: 309-312, 1989.

Wong, W.S.F. and Rahwan, R.G. : Anti ulcer activity of the calcium antagonist propyl-methylenedloxyindene. Pharmacologist. Abst. 149, 1989.

iv Wong, W.S.F. and Rahwan, R.G.: Antiulcer activity of the calcium antagonist propyl-methylenedioxyindene. I. Effect on cold-restraint stress-induced ulcers in rats. Gen. Pharmacol. 21: 321-325, 1990.

Wong, W.S.F. and Rahwan, R.G.: Antiulcer activity of the calcium antagonist propyl-methylenedioxyindene. II, Effects on acid secretion and gastric emptying in rats. Gen. Pharmacol. 21: 327-331, 1990.

FIELDS OF STUDY

Major Field: Pharmacology

Smooth Muscle Pharmacology - Dr. R.G. Rahwan

Pharmacology of Calcium - Dr. R.G. Rahwan

Gastrointestinal Pharmacology - Dr. R.G. Rahwan and D r. R.L. Stephens, Jr.

v TABLE OF CONTENTS

PAGE

DEDICATION ...... ii

ACKNOWLEDGMENTS ...... iil

VITA ...... Iv

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

CHAPTER I INTRODUCTION ...... 1

1.1 Anatomy of the Stomach and the Duodenum ...... 1

1.2 Histophysiology of the Gastric Glands ...... 12

1.3 Regulations of Gastric Acid Secretion ...... 17

1.4 Pathogenesis of Peptic Ulcer Disease ...... 26

1.4.1 Experimental models of gastric ulcers ...... 32

1.4.1.1 Cold-restraint stress ulcers...... 33

1.4.1.2 TRH-lnduced gastric ulcerations ...... 35

1.4.1.3 Cysteamlne-induced duodenal ulcers ...... 38

1.5 Pharmacology of Antiulcer Drugs ...... 41

1.5.1 Antisecretory agents ...... 41

1.5.1.1 Hz-receptor antagonists ...... 41

1.5.1.2 Antimuscarinic agents ...... 46

1.5.1.3 Anti gastrin agents ...... 49

vl 1.5.1.4 H+/K+-ATPase inhibitors ...... 51

1.5.1.5 A ntacids ...... 57

1.5.2 Mucosal protective agents ...... 58

1.5.2.1 Prostaglandins ...... 58

1.5.2.2 Sucralfate ...... 61

1.5.2.3 Colloidal bismuth subcitrate ...... 64

1.6 Calcium Antagonists and Peptic Ulcer Disease ...... 66

1.6.1 Calcium channel blockers ...... 67

1.6.2 Intracellular calcium antagonists ...... 71

1.6.3 Effects of calcium antagonists on experimental ulcers ...... 75

CHAPTER II RATIONALE AND AIMS OF THIS STUDY ...... 79

CHAPTER III ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE. EFFECTS ON COLD-RESTRAINT STRESS-INDUCED ULCERS, ACID SECRETION, MAST CELL DEGRANULATION AND GASTRIC EMPTYING IN RATS ...... 83

3.1 Introduction ...... 83

3.2 Methods and Materials ...... 86

3.2.1 Effect of pr-MDI on cold-restralnt stress-indi .ied ulcers ...... 86

3.2.2 Effect of pr-MDI on gastric acid secretion ...... 87

3.2.3 Effect of pr-MDI on mast cell degranulation ...... 88

3.2.4 Effect of pr-MDI on gastric emptying ...... 89

vii 3,2.5 Materials and data analysis ...... 91

3.3 Results ...... 92

3.3.1 Effect of pr-MDI on cold-restralnt stress-induced ulcers ...... 92

3.3.2 Effect of pr-MDI on gastric acid secretion ...... 96

3.3.3 Effect of pr-MDI on mast cell degranulation ...... 98

3.3.4 Effect of pr-MDI on gastric emptying ...... 101

3.4 Discussion ...... 101

CHAPTER IV ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE, EFFECT ON CYSTEAMINE-INDUCED DUODENAL ULCERS IN RATS ...... 112

4.1 Introduction ...... 112

4.2 Methods and Materials ...... 114

4.2.1 Cysteamine-induced duodenal ulcers ...... 114

4.2.2 Gastric acid secretion ...... 117

4.2.3 M aterials and data an n lysls ...... 118

4.3 Results ...... 119

4.4 Discussion ...... 125

CHAPTER V ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE. EFFECTS ON GASTRIC LESIONS IN RATS INDUCED BY COLD-RESTRAINT STRESS AND THYROTROPIN-RELEASING HORMONE ...... 132

5.1 Introduction ...... 132

viii 5.2 Methods and Materials ...... 134

5.2.1 Cold-restralnt stress ulcers ...... 134

5.2.2 TRH-induced gastric ulcerations ...... 135

5.2.3 TRH-induced gastric emptying ...... 135

5.2.4 TRH-induced gastric acid secretion ...... 136

5.2.5 Materials and data analysis ...... 137

5.3 Results ...... 138

5.3.1 Cold-restraint stress ulcers ...... 138

5.3.2 TRH-induced gastric ulcerations ...... 138

5.3.3 TRH-induced gastric emptying ...... 141

5.3.4 TRH-induced gastric acid secretion ...... 141

5.4 Discussion ...... 141

CHAPTER VI SUMMARY AND CONCLUSIONS ...... 148

REFERENCES ...... 153

ix LIST OF TABLES

TABLE PAGE

1 Effects of pr-MDI, verapnmll, and cimetidine on cold-restraint stress ulcer formation In rats ...... 94

2 Effects of pr-MDI, verapamil, and cimetidine on belhanecliol-induced gastric acid secretion ...... 97

3 Effect of pr-MDI and verapamil on mast cell counts at mucosal and submucosal area of the stomach of rats treated with 3.2 mg/kg bethanechol subcutaneously ...... 100

4 Effect of pr-MDI and verapamil on gastric emptying of rats treated with test meal for 1-hr ...... 102

5 Effects of drug pre-treatments on cysteamine- induced duodenal and gastric ulcers in rats ...... 120

6 Effect of cysteamine (425 mg/kg) on gastric acid secretion for 4 hours in pyloric-ligated rats ...... 121

7 Effects of drugs on bethanechol (3.2 mg/kg)- induced gastric acid secretion for 2 hours In pyloric-ligated rats ...... 122

8 Effects of pr-MDI on cold-restraint stress-induced gastric ulcers in rats ...... 139

9 Effect of pr-MDI on gastric erosions in rats induced by intracisternal administration of the Till I analogue, RX77368 140

10 Effect of pr-MDI on gastric emptying in rats treated intracisternally with the TRH analogue, RX77368 ...... 142

11 Effect of pr-MDI on gastric acid secretion in pyloric-Iignted rats treated intracisternally with the TRH analogue, RX77368 143

x LIST OF FIGURES

FIGURES PAGE

1 Anatomic regions of the stomach ...... 3

2 Schematic diagram of the muscularis propria of the stomach ... 5

3 Diagram of the arterial supply to the stomach ...... G

4 Schematic diagram of the enteric nervous system ...... 8

5 Anatomic regions of the duodenum in relation to the stomach and the pancreas (A), and diagram of the major arterial supply to the duodenum (B) ...... 11

6 Schematic diagram of an oxyntic gland in the body of the stomach ...... 14

7 Schematic diagram of a parietal cell with receptors for histamine, acetylcholine, and gastrin ...... 22

8 Diagramatic illustration of acute and chronic gastric mucosal lesions ...... 27

0 Schematic diagram showing the most common sites of peptic ulcers in man ...... 28

10 Schematic diagram describing the dynamic balance between the injurious factors and the protective mechanisms of normal gastric mucosa ...... 30

11 Schematic diagram showing the effects of the intracisternally-injected TRH on the function of the stomach ...... 36

12 Structures of the H 2 -receptor blockers ...... 43

13 Structures of antimuscarlnic agents ...... 47

14 Structures of antigastrin agents ...... 50

xi 15 Structures of the H*/K+-ATPase inhibitors ...... 54

16 Events leading to inhibition of gastric acid secretion by omeprazole analogue, timoprazole, within the parietal cell ...... 55

17 S tru ctu re of su cralfate ...... 62

18 Structures of the three major classes of calcium channel blockers ...... 68

19 Structures of some intracellular calcium antagonists ...... 72

20 The effect of pr-MDI on stress-induced ulcers in rats ...... 93

21 Photomicrographs of a section of the gastric mucosa (top) and the gastric submucosa (bottom), showing the metachromatically stained mast cells ...... 99

22 A relationship between % inhibition of stress-induced gastric ulcer and % inhibition of gastric emptying in rats b y pr-MDI ...... 103

xtl CHAPTER I

INTRODUCTION

1.1 Gross Anatomy of the Stomach and the Duodenum

The stomach is the most dilated segment of the entire gastrointestinal tract, which connects superiorly with the terminal portion of the esophagus at the esophago-cardlac orifice and inferiorly with the proximal duodenum at the pyloro-duodenal orifice. Stomach is a

J-shaped organ with a maximum storage capacity of 1500 ml in most normal individuals (Fenoglio-Preiser et al., 1989). It is located in the upper part of the abdomen, extending from the left hypochondrium into the epigastrium and the umbilicus. The functions of the stomach, which are constantly under neural and hormonal regulations, include gastric secretions of hydrochloric acid, protease pepsin, mucus, various hormones and electrolytes, food storage, and transformation of the ingested food into viscous mass called chyme for further digestion in the small intestine. In addition, high concentration of the gastric acid serves to sterilize the gastric contents, while the proteolytic pepsin enzyme initiates protein digestion.

Anatomic observations suggest that the stomach can be divided into

5 regions which are cardia, fundus, body (or corpus), antrum, and pylorus (Fenoglio-Preiser al., 1989) (Figure 1). The former 3 regions constitute the upper portion of the stomach which Is demarcated from the distal portion consisting of the antrum and the pylorus by a notchlike indentation, the inclsura angularis, on the lesser curvature side. Histological examinations show that the stomach is composed of 4 tissue layers: mucosa, submucosa, muscularis propria, and serosa

(McGulgan and Ament, 1989; Owen, 1986). When the stomach is empty, the gastric mucosal lining is thrown into numerous folds known as rugae, which consist of the mucosal and submucosal layers. The mucosal folds are flattened out In a distended or filled stomach. Rugae are most prominent in the fundus since stomach dilatation occurs in this region (Owen, 1986). Moreover, inumerable gastric pits (foveolae gastricae), representing invaginations of the mucosal epithelial lining into the lamina propria, can be identified throughout the surface of the gastric mucosa. Opening at the base of these pits are the gastric glands responsbile for gastric secretions. Pyloric mucosa has deep gastric pits which account for one half the thickness of the mucosa

(Fenoglio-Presier et al., 1989). Lamina propria of the mucosa is composed of connective tissues in which gastric glands, capillaries, lymphatic efferents, non-myelinated nerve fibers, and connective tissue cells such as mast cells and plasma cells are located (Owen, 1986).

Muscularis mocosa lies at the base of the gastric mucosa, which consists of outer longitudinal muscle layer and Inner circular muscle layers, together with some elastic fibers. Submucosa is made up of loose connective tissue with extensive arterial, lymphatic, and ganglionic 3

Esophagus

Fundus

C ardia

Lesser curvature

Body

Incisuro A ngularis Pylorus G reater curvature

A ntrum

Figure 1. Anatomic regions of the stomach. (Modified from McGuigan and Ament, 1989). plexuses localized in this tissue layer. The muscularis propria consists

of three spiral fibers oriented In 3 directions : the outer longitudinal

layer, inner circular layer, and Innermost obligue muscle fibers

(Fenoglio-Preiser et al., 1989) (Figure 2). Contraction of the circular

fibers constricts the lumen while contraction of the longitudinal fibers

compresses the vertical dimension of the stomach. At the distal end of

the pylorus, the Inner circular layer is greatly thickened to form the

pylorus sphincter (Owen, 1986). These three layers of smooth muscle

contain numerous receptors susceptible to neurohumoral regulation and

are responsible for gastric slow wave and burst contraction to facilitate

mixing of the ingested food into chyme and subsequent transport into

the duodenum (Meyer, 1990). The serosa is made up of dense

connective tissue lined by a layer of mesothellum.

The stomach is richly supplied with blood flow through arteries branched from the celiac, hepatic, and splenic arteries, which Include left and right gastric arteries, left and right gastroepiploic arteries, and short gastric artery (Gannon et al., 1984; McGuigan and Ament,

1989) (Figure 3). The arteries congregate in the submucosa to form extensive plexus, which give off arterioles and capillaries to the mucosa, muscularis propria, and serosa. The capillaries anastomose freely with the venules at the superficial lamina propria subjacent to the surface mucous cells (Gannon et al., 1984), and the blood is drained into the portal vein system in the liver. The gastric lymhatic drainage priclpally parallels the arterial supplies. Lymphatic plexuses are mainly localized in the muscularis mucosa and the submucosa 5

Longiludmnl musctc

Oblique gastric muscle fibers

C ircular muscle fibers

Figure 2. Schematic diagram of the muscularis propria of the stomach, showing the outer longitudinal layer, inner circular layer, and innermost obligue muscle fibers. (Adapted from Fenoglio- Preiser et al., 1989). 6

Short gastric Afs

Lt gastric A.

Celiac A.

Hepatic A.

Rt. gastric A. Lt gastro­ epiploic A. G astro- — Splenic A. duodenal A.

Rt. gastro­ epiploic A.

Figure 3. Diagram of the arterial supply to the stomach. (Adapted from Guth and Leung, 1987). sending out lymphatic efferents to the superficial gastric mucosa (Owen,

1986).

The stomach is innervated by both the sympathetic and

parasympathetic divisions of the autonomic nervous system, and the

enteric nervous system (Cooke, 1986; Fenoglio-Preiser et al., 1989).

The preganglionic fibers of the sympathetic nerve trunk arise from 7th and 8th thoracic segments of the spinal cord and concentrate in the

celiac plexus from which the postganglionic fibers project to the stomach wall and regulate the functions of the target organ (Fenoglio-Preiser et al., 1989). The parasympathetic fibers are derived from the left and right vagus nerves, and terminate at the gastric submucosal ganglionic cells (Melssner's plexus) and the myenteric plexus (Auerbach's Plexus) in the muscularis propria (Cooke, 1986; Owen, 1986). The postganglionic fibers of these plexuses extend to the secretory and motor components of the stomach and regulate their activities.

Activation of the parasympathetic nervous system usually increases

gastric secretions from the exocrine and endocrine glands, gastric mucosal blood flow, and gastric motility; to the contrary, activation of the sympathetic nervous system reverses these effects. The enteric nervous system, which consists of extensive integrative interneurons throughout the Melssner's and myenteric plexuses, can mediate reflex activity of the stomach independent of the regulation by the central nervous system (Cooke, 1986) (Figure 4). The sensory neurons detect the mechanical, chemical, and thermal conditions of the gastric lumen and, through the information processing and integration among the 8

Epithelium Epithelial receptors

Epithelialf receptors Blood vessel Sensory Motor neuron neuron

Intemeuron Neural receptor

Figure 4. Schematic diagram of the enteric nervous system, showing the interactions of sonory neurons and motor neurons with interneurons, epithelial cells, and portion of the muscularis mucosa. (Adapted from Fenoglio-Preiser et al., 1989). interneurons, specific motor neurons are activated and neurotransmitters are released from the nerve terminals to the mucosal effectors in the epithelium, muscularis mucosa, and blood vessels

(Cooke, 1986), In addition to the presence of the neurotransmitters such as norepinephrine and acetylcholine, many peptide hormones including vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrin releasing peptide (GRP), bombesin, substance

P (SP), enkephalin, somatostatin, gastrin, cholecystokinin (CCK), and neuropeptide Y (NPY) are also found in the neurons of the gastric wall, gastric mucosa, and nerve plexuses (Fenoglio-Preiser et al., 1989;

Owen, 1986).

The duodenum begins at the pyloro-duodenal junction and extends for 25 cm In length forming a C-shaped curve which encloses in its concavity the head of the pancreas (Figure 5A). The duodenal wall is composed of mucosa, submucosa, muscularis propria, and serosa. Apart from the mucosa, these tissue layers are structurally similar to those of the stomach. The proximal duodenum Is frequently exposed to acidic chyme of extreme pH emptied from the stomach and one of its functions is to neutralize these gastric contents by producing enough HCOa“ from the duodenal mucosal epithelium (Flemstrom, 1987; Isenberg et al.,

1986). Moreover, the Brunner's glands located primarily In the submucosa of the proximal duodenum constantly secrete alkaline mucus of pH 8.2-9,3 in response to acid to assist removal of H* in this region

(McGuigan and Ament, 1989 Fenoglio-Preiser etal., 1989). On the other hand, pancreatic and biliary alkaline secretions gain entry into the 10

duodenum within 10 cm distal from the pylorus through the main and

accessory pancreatic ducts, which are transported retrogradely against

the peristaltic wave to the proximal duodenum to enhance acid

neutralization (McGuigan and Ament, 1989). Other two main functions of

the duodenum are digestion of chyme by pancreatic and Intestinal

enzymes, and absorption of nutrients by the epithelial absorptive cell.

The duodenal mucosa is lined by columnar epithelium, which is thrown

into permanent crescentic folds, known as plica circulares, with villi

projecting into the intestinal lumen (Fenoglio-Preiser etal., 1989; Gray* e t al., 1989). The villi contain central lacteal, a lymphatic vessel, capillaries, and nerve fibers, and are lined with epithelial absorptive cells covered wtili microvilli to increase surface, ore for digestion and absorption. Detween the villi are openings of simple tubular glands, known as crypts of Lieberkuhn, located in the lamina propria, which, in turn, connect with the Brunner's glands located in the submucosa

(G ray et al., 1989).

The proximal duodenum is richly supplied with capillaries and arterioles branched from the supraduodenal artery and the posterior superior pnncreaticoduodenal branch of the gastroduodenal artery, which, in turn, are derived from the common hepatic artery (Figure

5B). The remaining parts of the duodenum are supplied by anterior and posterior inferior pancreaticoduodenal arteries branched from the superior mesenteric artery (Skandalakls et al., 1989; Fenoglio-Preiser et al., 1989). The arterial blood supply is drained by suprapyloric veins which open to the portal vein or the posterior superior II

(A) Common bile duct

Accessory poncreolic duc( (Sonlorini) "Main pancreolic _ _ duct ff CWirsungJ\

(B)

Supraduodenal a.

Gastroduodenal a.

Pancraalico- duodenal arches

Las) duodenal branch

Figure 5. (A) Anatomic regions of the duodenum In relation to the stomach and the pancreas. (B) Major arterial supply to the duodenum. SMA stands for superior mesenteric artery. (Adapted from Gray et al., 1G89 and Skandalakis et al., 1989, respectively). pancreaticoduodenal vein (Skandalakis et al., 1989), The duodenum is also richly supplied with lymphatic capillaries known as lacteals which form the Peyer's patches in the lamina propria and the submucosa. The nervous innervations of the duodenum are relatively similar to that of the stomach, in that both Meissner's plexus and myenteric plexus can be found in the submucosa and the muscularis propria, respectively.

The sympathetic postganglionic fibers regulating the functions of the duodenum are derived from the celiac and superior mesenteric plexus

(Skandalakis et al., 1989).

1.2 Histophysiology of the Gastric Glands

The entire surface of the gastric mucosa is lined by a layer of tightly joined simple columnar epithelium of surface mucous cells. The gastric mucosa is interrupted by numerous gastric pits penetrating into the lamina propria and the pits are covered by the same epithelial cells.

At the bottom of the gastric pits are the branched tubular gastric glands, and their secretory products are excreted into the gastric lumen through these foveolae. It has been estimated that there are 3.5 million foveolae serving 15 million gastric glands in a human stomach

(McGuigan and Ament, 1989). The surface mucous cells are densely packed with granules and secrete neutral polysaccharide protein called mucin which forms a thick layer of mucus covering the superficial epithelium to protect the mucosa from extreme intragastric pH and proteolytic pepsin enzyme, and to lubricate the luminal surface to facilitate transport of gastric contents. In addition, this mucus gel 13

layer functions as a pH gradient between the intraluminal side of pH

1-2 and the superficial mucosal side of pH 7 (Owen, 1986; Richardson,

1989). Inorganic HCO 3 ” is also continuously secreted from these cells

to help in neutralizing the H+ back diffused from the lumen

(Richardson, 1989). Other studies suggest that H C03~, released from

the parietal cells located in the deeper part of the gastric mucosa (see

below), is transported by the capillaries to the superficial lamina

propria right beneath the basement membrane of the mucous cells and is

diffused across these cells into the gastric lumen to assist neutralization

of the H* (Gannon et al., 1984). The surface mucous cells are

completely renewed every 4-8 days (Fenoglio-Preiser et al., 1989), and

without this mucus gel layer, mucosal ulceration occurs.

The gastric mucosa contains three distinct types of secretory

glands: caridac, pyloric, and oxyntic glands. The former two mainly

secrete mucus and lysozyme into the gastric lumen and are located correspondingly in the lamina propria of the cardiac and pyloric-antrum regions of the stomach. The oxyntic glands make up most of the

gastric mucosa of the fundus and the body of the stoamch. These branched tubular glands can be divided Into sections such that the upper isthmus portion contains undifferentiated cells and mucous neck cells, the midsection consists of parietal cells, and the base of these glands is mainly composed of chief (zymogen) cells, enteroendocrlne cells, and somatostatin-containing cells termed as 'D cells1 (McGuigan and Ament, 1989; Wolfe and Soil, 1988) (Figure 6). 14

Surface mucous colls

0

Mucous neck cells

Parietal cell

0

Somatostatin e cell

9 Endocrine cell

Chief cells

Schematic diagram of an oxyntic gland in the body of the F igure 6 stomach. (Adapted from Wolfe and Soil, 1988). 15

The undifferentiated cells actively undergo proliferation and differentiation. They move upward to replace the surface mucous cells and migrate deeper to renew parietal cells, chief cells, and enteroendocrlne cells. The mucous neck cells secrete transparent acidic mucus rich in glycosaminoglycan. Parietal cells are mainly localized in the upper half of the oxyntic gland and also interspersed with chief cells in the lower half of the glands. Parietal cells are large (20-35 um in diameter) and pyramidal in shape. A normal human stomach contains approximately 1 billion parietal cells and they are renewed every 1-3 years (Owen, 1986; Soli, 1989a). These cells are responsible for hydrochloric acid secretion in the stomach and distinctly featured by the extensive intracellular canaliculi formation lined with elongated microvilli at the apical membrane upon stimulation. Under resting or nonsecretory condition, the intracellular canaliculi collapse and are transformed into closed membrane systems of tuboveslcular structures in the cytoplasm adjacent to the apical cell membrane (Forte and Wolosin,

1987; Sachs et al., 1988). Hydrochloric ncid secretion by the parietal cell is an energy dependent active transport process via activation of the H*/K*-ATPase located in the microvilli of the intracellular canaliculi at the apical membrane. This energy-dependent process is partly reflected ultrastructurally by the abundant number of mitochondria, which constitute 34% of the cell volume, in the cytoplasm of the parietal cell (Sachs, 1987). In addition, the parietal cells are the site of synthesis and release of the intrinsic factor, a glycoprotein, which avidly binds to vitamin B 12 to form a complex necessary for the 16

absorption of the vitamin B 12 by the intestinal cells in the ileum

(Fenoglio-Preiser et al., 1989; Sanjiv, 1989). Chief cells are generally

distributed in the lower half of the glands and are responsible for the

release of proenzyme pepsinogen I and II from the zymogenic granules

by exocytosis. The inactive enzymes will be converted to active

proteolytic enzyme pepsin under acidic environment. The

enteroendocrine cells are located principally at the base of the oxyntic

gland and are filled with basally located hormone-containing small

granules which can be stained with silver salts or chromic-acid

containing fixatives. If the cells can be impregnated with the chromic-

acid, they are then called enterochromaffln cells or enterochromaffin­

like cells, which usually contain serotonin, histamine, motilin, and

substance P (Fenoglio-Preiser, 1989). The function of these cells has

not been established; however, they are involved in pathological

conditions such as enterochromaffin-like cells hyperplasia and gastric

carcinoid which are associated with hypergastrinemia in patients with

pernicious anemia due to chronic atrophic gastritis and Zolllnger-Ellison

syndrome due to pancreatic gastrinoma, or in experimental animals on

long-term treatment with potent anti-secretory agents (Coupe et al.,

1990; Creutzfeldt, 1988; Harvey et al., 1985). D cells are primarily

located at the base of the oxyntic glands. These somatostatin-

containing cells display long cytoplasmic processes reaching to other effector cells such as G-cells (see below), parietal cells, chief cells,

and enteroendocrine cells, to exert their local modulatory (paracrine) action (McGuigan and Ament, 1989). In most cases, somatostatin acts 17

to inhibit the function of its target cell via activation of the inhibitory

G protein which attenuates the adenylate cyclase activity and

subsequent cAMP formation (Lucey and Yamada, 1989).

Pyloric glands are branched and extensively coiled secretory

glands located beneath the openings at the bases of the long gastric

pits. They contains mucus-secreting cells which are similar to the

mucous neck cells in the oxyntic gland (Ito, 1987). In addition, these

glandular cells also produce large amount of lysozyme (Fenoglio-Preiser et al., 1989). Gastrin, the most potent hormonal secretagogue, Is

produced by the endocrine G cell located in the middle to deeper portion of the pyloric gland. The G cell has a narrow apical surface covered with long microvilli serving as sensory receptors to regulate the release of the hormone upon stimulation by increases in intragastric pH and by chemical substances such as protein and amino acid (Ito,

1987; Wolfe and Soil, 1988). Gastrin is released at the base of the cell by exocytosis into the extracellular space and is diffused into the nearby capillaries to exert its endocrine action on the target cells. In addition to its stimulatory effect on gastric acid secretion, it exhibits trophic effect on the gastric mucosa resulting in a condition known as enterochromaf fin-Ilke cell hyperplasia, which is primarily due to hypergastrinemia (Coupe et al., 1990; Hakanson and Sunder, 1986).

1.3 Regulations of Gastric Acid Secretion

Hydrochloric acid secretion from the parietal cell of the gastric mucosa is primarily regulated by three major pathways which include 18

neurocrine action of acetylcholine, endocrine effect of gastrin, and

paracrine function of histamine (Bertaccini and Coruzzl, 1988; Soil and

Berglindh, 1987; Wolfe and Soli, 1988). These three secretagouges activate their respective receptors, namely, the muscarinic receptor,

histamlnergic receptor and gastrinergic receptor, located on the

basolateral membrane of the parietal cell to stimulate gastric acid

secretion. Acetylcholine is released from the varicosities of the postganglionic, parasympathetic fibers derived from the submucosal and myenteric plexuses upon stimulation by the thought, sight, smell or taste of appetizing food, and by gastric distention, which correspond to the cephalic phase and gastric phase of the gastric acid secretion

(Feldman, 1984; Wolfe and Soli, 1988). In vitro studies using isolated parietal cells have demonstrated that the muscarinic receptor located on the basolateral membrane exhibited 100-fold higher affinity for atropine, a nonselective muscarinic antagonist, than for pirenzepine, an Mi- selectlve antagonist (Chan and Soil, 1988; Feldman, 1984; Hammer and

GiachetU, 1982). Functionally, atropine was 100-fold more potent than pirenzepine in inhibiting carbachol-induced acid secretion from the isolated parietal cells as measured by the accumulation of1

Mi-selective agonist, in pithed rats (Hammer and Giachetti, 1982).

Based upon these pharmacophysiologie evidence, it has been concluded that cholinergic regulation of gastric acid secretion is mediated by Ma- receptors located on the parietal cell and Mi-receptors situated in the submucosal and myenteric plexuses (Bertaccini and Coruzzl, 1988;

Feldman, 1984). Furthermore, Ma-receptors have also been Identified on the plasmalemma of other cells of the gastric mucosa such as chief, somatostatin, gastrin, glucagon, mast, enterochromaffin-like cells (Chan and Soil, 1988). Therefore, gastric acid secretion may be indirectly regulated by the cholinergic pathway through the releases of these hormones and polypeptides.

Gastrin is released from the G cell located in the pyloric mucosa and intestinal mucosa during the gastric phase and the intestinal phase of the gastric acid secretion (Wolfe and Soli, 1988). The release of gastrin is initiated by the activation of the Ma-receptor on the G cell membrane by cholinomimetics (Chan and Soli, 1988; Chan et al., 1988), by the chemical stimulants In food such as protein and amino acids, especially tryptophan and phenylalanine (Richardson, 1988; Sanjiv, 20

1989), by calcium and magnesium (Richardson, 1988), and by Increase in intraluminal pH above 3.0 to 3.5 (Decktor et al., 1989; Wolfe and

Soil, 1988). The released gastrin can directly act on the gastrin receptor on the parietal cell to stimulate gastric acid secretion, which can be blocked competitively by the selective gastrin antagonist, proglumide (Bertaccini and Coruzzi, 1988). Moreover, it has been proposed that gastrin can induce an indirect effect on acid secretion by releasing histamine from the histamine-secreting cells such as the mast cell and the enterochromaffin-like cell, in which gastrin receptors have been found on the plasma membrane (Black and Shankley, 1987; Waldum and Sandvik, 19B9).

The sources of histamine for paracrine stimulation of acid secretion from the parietal cell has been an unresolved issue. Many studies have shown that, in rat and in rabbit, the predominant stores of fundic mucosal histamine are contained in enterochromaffln-like cells. In contrast, in dog and In human, histamine is stored in the mast cell

(Chan and Soli, 1988; Soli et al., 1981; Waldum and Sandvik, 1989).

However, other studies suggested that stress-induced mast cell degranulation releases large amounts of histamine in rats, which Is the main cause of stress-ulceration (Cho et al., 1985; Ogle et al., 1985).

These histaminocytes, histamine-secreting cells, express muscarinic M2- and gastrin-receptors, and the bindings of these receptors with acetylcholine and gastrin, respectively, has been shown to release histamine. The released histamine then activates hydrochloric acid secretion from the gastric parietal cell by interacting with the 21

histaminergic receptor which has been pharmacologically characterized as

Hz-receptor since it is competitively blocked by the H 2 antagonists

(Black et al., 1972; Black and Shankley, 1987). Although these three distinct pathways have been identified and characterized, their relative contributions to acid secretion has not been established. An additional factor complicating the elucidation of the relative effect on acid secretion by each secretagogue is the interdependency among these pathways in regulating acid secretion (Wolfe and Soli, 1988; Feldman,

1984). It has been reported that potentiating effects on oxygen consumption and aminopyrine accumulation in isolated parietal cells existed between histamine and botli carbachol and gastrin (Soli and

Berglindh, 1987). Moreover, it has been observed that H 2-recep tor antagonist or muscarinic antagonist reduced acid secretion stimulated by all three secretagogues (Feldman, 1984; Soli and Berglindh, 1987; Wolfe and SoU, 1988).

Activations of these receptors initiate sequence of events from

Increases in intracellular second messengers, activations of protein kinases, phosphorylation of regulatory proteins, morphological transformations, to H* secretion from the gastric parietal cells (Figure

7). It has been clearly demonstrated that histamine stimulates intracellular adenosine S'jS'-monophosphate (cAMP) production In isolated parietal cells via H 2-receptor activation coupled to enhanced activity of the catalytic subunit of the adenylate cyclase system in the basolateral membrane (Soil and Berglindh, 1907; Wolfe and Soli, 1988).

The intracellular cAMP protein kinase which, in turn, phosphorylates • Histamine-2 Receptor Basolateral Membrane

Adenylate cyclase + GTP Nerve tcAMP

Muscarinic - 2 Receptor lor Protein Acetylcholine Kinase Ca++ Calmodulin Stood Vessel ■ ■ B Gastrin Receptor

Apical Membrane

Figure 7. Schematic diagram of a pnrletnl cell with receptors for histamine, acetylchlollne, and gastrin. Activation of these receptors elevates intracellular Ca*2 and cAMP concentrations, which, in turn, stimulate HC1 secretion by the H+/K*-ATPase, (Adnpted from Feldman, 1909). 23

protein kinase which, in turn, phosphorylates specific target proteins

and ultimately leads to H* secretion from the parietal cell (Beracclnl and

Coruzzl, 1988; Forte and Wolosln, 1987). The close relationship between

enhancement of cAMP production by histamine and stimulation of H+

secretion has been confirmed by the observation that dibutryl-cAMP

(bdcAMP), an analogue of cAMP, lsobutylmethylxanthine (IBMX), a

phosphodiesterase inhibitor, and forskolin, an activator of adenylate

cyclase, increase acid secretion from the Isolated parietal cells

(Negulescu and Machen, 1988; Soil and Berglindh, 1987).

In contrast, cholinergic and gastrinergic activations enhance intracellular calcium concentration in isolated gastric glands and parietal cells (Chew and Brown, 1986; Negulescu and Machen, 1988). Muallem and Sachs (1985) reported that carbachol elevated intracellular calcium concentration in Isolated parietal cells by enhancing membrane permeability to extracellular Ca+2 without calcium release from the intracellular stores, and the calcium Influx could be blocked by lanthanum. To the contrary, other studies demonstrated that carbachol- and gastrin-induced elevations In intracellular calcium concentration are mediated by calcium mobilization from the internal stores via stimulation by inositol 1,4,5-trisphosphate (IPs) which Is formed by phospholjpase

C hydrolysis of phosphatidylinositol 4,5-bisphosphate (Chew et al.,

1986; Chew and Brown, 1986), and the increases in cytosolic calcium concentration could not be blocked by organic calcium channel blockers such as nicardipine, verapamil, or nifedipine (Negulescu and Machen,

1988; Chew and Brown, 1986; Wolfe and Soli, 1988). The Increased 24

cytosolic calcium activates calmodulin which, in turn, phosphorylates

unidentified proteins and finally stimulates H* secretion from the

parietal cell (Black et al., 1989; Forte and Wolosin, 1987). Recently,

many elaborate studies have demonstrated that histamine induces

increase in intracellular calcium concentration in isolated gastric glands

and parietal cells via mobilization of calcium from the internal stores,

which is not mediated by the enhanced formation of IP 3 (Chew and

Brown, 1986 Negulescu and Machen, 1988). Since there was a definite

lag in the rise in cytosolic calcium concentration following stimulation by

histamine and forskolin compared with carbachol, the authors suggested

that histamine-stimulated increase in cellular cAMP mediates the rise in

intracellular calcium concentration by an unknown mechanism (Chew,

1986; Negulescu and Machen, 1908). However, studies showed that dbcAMP alone or in combination with IBMX stimulate acid secretion without altering the intracellular calcium concentration, suggesting that an increase in cytosolic calcium concentration does not play an obligatory role in histamine-induced activation of parietal cells (Waldum and Sandvik, 1989).

Apart from the three well-characterized excitatory receptors, a new receptor for prostaglandin Ea (PGE2) has been identified on the membrane of the isolated parietal cells (Bertaccini and Coruzzl, 1988;

Wolfe and Soli, 1988). Active' ion of this receptor results in inhibition of acid secretion in human. In vitro studies using isolated parietal cells showed that PGEa inhibited histamine-stimulated acid secretion from the parietal cell by blocking the cAMP production via activation of the 25

inhibitory G protein and subsequent inhibition of adenylate cyclase

activity (Chen et al., 1988; Wolfe and Soli, 1988). Other endogenous

regulators of acid secretion in the fundic mucosa include the stimulatory

peptides such as bombesin and cholecystoklnin, and the inhibitory

compounds such as somatostatin, epidermal growth factor, vasopressin,

and enteroglucagon (Bertaccini and Coruzzi, 1988; Soli and Berglindh,

1987).

The biochemical events subsequent to the activations of cAMP-

dependent protein kinase and calmodulin, and prior to the stimulation of

H+/K+~ATPase activity are not fully understood. However, elevations of

cAMP and intracellular calcium concentration in the parietal cell have

been shown to induce transformation of the tubulovesicular structures

in the cytoplasm into secretory canaliculi covered with elongated microvilli at the apical membrane, on which the enzyme H*/K*-ATPase is located (Forte and Wolosln, 1987; Sachs et al., 19BB). Moreover, activation of this membrane-bound enzyme is associated with a concomitant increases in K* and Cl" conductances at the apical membrane resulting in the formation of HC1 (Forte and Wolosin, 1987).

Studies on the H+/K+-ATPase activity in gastric microsomal vesicle isolated from the parietal cell demonstrated that luminal K+ is an absolute requirement for the operation of the H* pump driven by the energy generated from the hydrolysis of ATP. The Hf/K*-ATPase is a countertransport pump, electroneutrally exchanging cytosolic H+ for luminal K* (Forte and Wolosin, 1987; Sachs, 1987). In order to maintain ionic balance within the acid-secreting parietal cells, K+ and 26

Cl” homeostases are regulated by transport pathways on the basolateral

m em brane.

1.4 Pathogenesis of Peptic Ulcer Disease

Peptic ulcer is defined as mucosal lesion of the upper

gastrointestinal tract penetrating through the muscularis mucosa into

the submucosa. In contrast, mucosal damage that does not extend through the muscularis mucosa is considered as superficial erosion

(Richardson, 1989; Sanjiv, 1989) (Figure 8). Peptic ulcers usually occur at regions frequently exposed to extreme pH, such as the lower esophagus, stomach, and proximal duodenum, and occasionally, at postbulbar duodenum and Jejunum (Richardson, 1989). It Is estimated that 5-10% of the U.S. population will develop symptomatic peptic ulcer disease In their life time. Each year in the United State, 4 million

Americans suffer from active ulcers, and 350,000 new cases are being diagnosed (Richardson, 1989; Sanjiv, 1989). Duodenal ulcer is 4 times more common than gastric ulcer in the United States, whereas, gastric ulcer is 5-10 times more common than duodenal ulcer in Japan (Carlstedt and Stanaszek, 1988; Richardson, 1989). The most common site of gastric ulcers are located near the incisure' angularls along the lesser curvature and at the antrum region, while the most common site of duodenal ulcer is located on the anterior antimesenteric wall of the proximal duodenum (Richardson, 1989) (Figure 9). Although peptic ulcer disease is a very common and well-characterized health problem, the pathogenesis of this upper gastrointestinal disorder has yet to be 27

Erosion Acute ulcer Chronic ulcer

Mucosa ------► //Ziit////// Submucosa — Muscularis— ► S erosa ------► Sclerosis

Figure 8. Diagramatic illustration of acute and chronic gastric mucosal lesions. (Adapted from Brooks, 1985). 28

PEPTIC ULCERS Peptic ulcer disease Is a major health problem In the U.S. that strikes up to t In 13 Americans at some point In fife. Peptic ulcer disease is an umbrella term that Includes duodenal ulcer (found In the lop ot the small Intestine) and gastric (stomach) ulcer. The Illustrations below show the most common sites ol these ulcers.

Most common site of the DUODENAL ulcer (4 Umes more common than gastric ulcer)

Stomach

Most common sites of GASTRIC ulcer

Figure 9. Schematic diagram showing the most common sites of peptic ulcers in man. (Adapted from Carlstedt and Stnnaszek, 1988). 29

determined. In general, it is thought that peptic ulcer disease signifies

a breakdown of balance between the luminal aggressive factors and the

mucosal defense mechanisms. The mucosal damaging factors include

hydrochloric acid, proteolytic pepsin enzyme, bile, and pancreatic

enzymes, while the mucosal protective elements encompass secretions of

mucus and HCO 3 , mucosal blood flow, cell regeneration, and endogenous

prostaglandin (Sanjiv, 1989; Soli 1989) (Figure 10).

Gastric acid hypersecretion occurs In (25-50)% of the patients with

duodenal ulcers. The rise in acid secretion might be associated with increased parietal cell mass which is reflected by increased maximal acid output in response to pentagastrin or histamine as compared to healthy subjects (Soli, 1989), Duodenal ulcer patients also exhibit enhanced basal acid output independent of any increase in fundic parietal cell mass, which has been shown to be mediated by heightened basal vagal tone (Kohn et al., 1985). Other physiologic abnormalities that precipitate duodenal ulcers include antral G cell hyperfunction, impaired acid-Inhibition of gastrin release, Zollinger-Ellison Syndrome, and enhanced sensitivity of the parietal cell to gastrin (Soil, 1989). In addition, rapid gastric emptying of liquid and solid meals in duodenal ulcer patients has been postulated as a possible pathogenetic factor causing duodenal mucosal acid overload that exceed the compensatory m ucus and HCOj' secretions from the superficial mucous cells and duodenal glands (Soli, 1989; Lam et al., 1982), On the other hand, recent findings revealed that a subset of duodenal ulcer Is genetically predisposed from autosomal dominant Inheritance of hyperpepsinogenemia 30

Attack

Pepsin Hydrochloric acid Bile acids Other (e.g. C. Pylori?)

Mucosa

Muscularis _ mucosa Submucosa - ® .

Muscle layer

Serosa ------Iff! Mucosal Mucus Blood Endogenous Bicarbonate Cell barrier flow prostaglandins restitution

Defense

Figure 10. Schematic diagram describing the dynamic balance between the injurious factors and the protective mechanisms of normal gastric mucosa. (Adapted from Chopra, 1989). 31

I, which has been shown to reflect increases in fundie parietal cell mass and gastric acid secretion (Richardson, 1989; Soil, 1989), In addition, individuals inherited with multiple endocrine neoplasm syndrome

(MEN)-type I develop duodenal ulcer because of islet cell gastrinoma, which secrete an excessive amount of gastrin resulting in acid hypersecretion. Since (50-75)% of the duodenal ulcer patients secrete gastric acid within the normal range, this indicates that local disruption of mucosal defense mechanism might play a permissive role for duodenal ulcer formation induced by adequate amount of acid and pepsin. It has been demonstrated that duodenal ulcer patients have decreased proximal duodenal mucosal HCOj- production at rest and in response to HC1, which predisposes the duodenum to mucosal damage by extreme pH

(Isenberg et al., 1987). Several lines of evidence suggested that duodenitis and antral gastritis associated with infection by helicobacter pylori, previously known as Campylobacter pylori, are major predisposing causes of duodenal ulcer (Goodwin, 1988; Soli, 1989).

In gastric ulcer, on the other hand, patients often secrete acid and pepsin within or lower than the normal range. A hypothesis that the diminished level of acid secretion in gastric ulcer patients might be due to increased H+ back diffusion into the ulcerated gastric mucosa remains to be established (Silen, 1988). It has been proposed that a defective pyloric sphincter is an important pathogenetic factor leading to gastric ulcer, which allows duodenal contents such as bile acid, lysoleclthln, and pancreatic enzymes to reflux Into the stomach to damage the gastric mucosa resulting in gastritis and ulceration (Boyle et al., 1984; Richardson, 1989). Other studies demonstrated that delayed gastric emptying in Individuals with gastric ulcer due to antral hypomotility results in prolonged food retention, increased gastrin release, increased acid secretion, and subsequent gastric ulceration

(Richardson, 1989). On the other hand, chronic superficial and atrophic gastritis are frequent findings in patients with gastric ulcers.

Substantial evidence demonstrated that prostaglandins of the E series exert mucosal protective actions which include increased mucosal blood flow, HCOj” and mucus secretion, and cell renewal (Miller, 1983).

Long-term administration of aspirin or other nonsteroidal anti­ inflammatory drugs, which inhibit the action of cyclo-oxygenase and the subsequent formation of PGE, disrupt the mucosal barrier and produce gastric ulceration (Graham et al., 1988; Roth and Bennett, 1987).

1.4.1 Experimental models of gastric ulcers

Despite intensive investigations, the pathogenetic mechanisms underlying peptic ulcer disease In humans Is still poorly understood.

Since chronic gastric ulcer models are much more difficult to produce, which often require excision of large segment of gastric mucosa or injection of acid into the serosal tissue of the stomach, major findings regarding the biological mechanisms of ulcerogenesis and anti-ulcer drug assessments have been derived from the studies on acute gastric ulcer models in rats (Silen, 1988), Acute gastric ulcer models include pyloric- ligation (Shay et al., 1945; Dal and Ogle, 1974); restraint stress (Pare and Glavin, 198G); cold-restralnt stress (Senay and Levine, 1967;

Glavin, 1988), hemorrhagic shock (Silen, 1988), central administration 33 of thyrotropin-releasing hormone (TRH) (Nakane et al., 1985; Goto and

Tache, 1985), oral administrations of ethanol (Cho et al., 1989;

Ghanayem et al., 1987) and non-steroid anti-inflammatory drugs (Hertz and Clorec, 1989; Ghanayem et al., 1987), Intra-arterial administrations of endothelin (Whittle and Esplugues, 1988), platelet-activating factor

(Esplugues and Whittle, 1988a), and thromboxane A2 (Esplugues and

Whittle, 1988b). Among these models, the superficial gastric mucosal ulcerations produced by cold-restraint stress and hemorrhagic shock highly resemble the stress ulcers in patients with severe trauma or serious medical illness (Silen, 1988). The frequency of occurrence of stress ulcers in seriously ill patients can be as high as 100% with mortality rate approaching 50% when massive bleeding becomes manifest

(Kleiman et al., 1988).

1.4.1.1 Cold -re s train t stress ulcers

It is generally accepted that gastric mucosal ischemia is the primary factor leading to stress ulcers. In experimental models, gastric mucosal blood flow is significantly reduced In rats subjected to cold- restraint stress (Robert et al., 1989; Murakami et al., 1985). The reduction of mucosal blood flow has been shown to be associated with concomitant increase in blood viscosity (Murakami et al., 1985). On the other hand, it has been demonstrated that cold-restraint stress induces high amplitude, long duration gastric muscular contraction mediated through Hi-receptor activation by histamine released from the mast cell and muscarinic receptor activation, which, in turn, mechanically 34

constricts mucosal microvasculatures resulting in ischemia and ultimately

mucosal necrosis (Cho et al., 1985; Garrick et al., 1986a,b). It w as,

therefore, suggested by Garrick et al. (1986a) that gastric

hypermotility plays a major role In cold-restraint stress ulcers in rats.

Recent findings showed that intra-arterlal infusions of endothelinfan

endothelium-derived peptide (Whittle and Esplugues, 1988)],

thromboxane A i and its analogues (Esplugues and Whittle, 1988a), or

platelet-activating factor (Esplugues and Whittle, 1988b) into the left

gastric artery produce substantial gastric mucosal damage resulting from

microvascular congestion and stasis of mucosal blood flow. These

endogenous pro-inflammatory mediators are released locally and have

been suggested as possible contributing factors to gastric erosions

induced by cold-restraint stress and hemorrhagic shock.

Many studies suggested that luminal acid is not associated with

cold-restraint stress-induced gastric ulceration since total neutralization

of luminal acid by NaHCOj did not prevent ulcer formation (Cho et al.,

1985; Dai and Ogle, 1974), and, In many instances, acid secretion was

significantly suppressed (Dai and Ogle, 1974; Garrick et al., 1986).

The reduction of acid secretion might be a consequence of decreased

gastric mucosal blood flow. Moreover, it has been demonstrated that

diltiazem, a calcium channel blocker, did not influence gastric acid

secretion but reduced stress gastric lesion formation (Glavin et al.,

1989). However, it has been argued that acid is a prerequisite for the

development of mucosal injury because ulceration does not occur with a luminal pH>7 (Klelman et al., 1988; Silen, 1988). The controversy 35 regarding luminal H* back diffusion into the mucosal tissue resulting in mucosal damage requires further clarification (Silen, 1988). Other possible mechanisms documented to contribute to cold-restraint stress ulcers in rats Include reduction of HCOj", mucus and PGE2 productions, and formation of oxygen-derived free radicals during reperfusion after mucosal ischemia (Kleiman et al., 1988; Silen, 1988).

1.4.1.2 TRH-induced gastric ulcerations

Intraclsternal microinjection of thyrotropin-releasing hormone

(TRH), or its stable analogue, RX77368, dose-dependently elicits gastric ulcerations in the glandular portion of the stomach in rats within 2-4 hrs (Goto and Tahce, 1985; Nakane et al., 1985) (Figure

11). The failure of intravenous infusion of the neuropeptide to produce gastric erosions in rats indicates that the TRH-induced gastric ulceration is mediated by central nervous system activation. It has been reported that TRH given centrnliy stimulated vagal efferent discharge as measured by attaching bipolar electrodes to the cervical and gastric vagus nerves (Somiya and Tonoue, 1984; Tache et al., 1985), and the subsequent ulcer formation was blocked by antisecretory doses of atropine, cimetidlne, and omeprazole (Goto and Tache, 1985). These findings suggest that gastric erosions produced by central TRH is associated with enhanced parasympathetic outflow and acid hypersecretion. The mechanisms underlying acid response to central vagal stimulation by TRH involve activation of muscarinic receptors directly at the parietal cell and indirectly through the release of 36 TRH

RAT BRAIN BRAIN

ATSH AProlactin AParasympathetic \ outflow

AGastric Acid Pepsin GMBF Emptying Motility Ulcers

STOMACH

Figure 11. Schematic diagram showing the effects of the intracisternally-lnjected TRH on the functions of the stom ach . 37

histamine. By contrast, gastrin does not appear to participate in TRH-

induced gastric acid secretion (Yanagisawa etal., 1990).

Aisde from gastric acid hypersecretion, other possible mechanisms

mediating TRH-induced gastric ulcerations in rats include elevations of

gastric papsin secretion (Tache et al., 1989a), gastric mucosal blood

flow (Thiefln et al., 1989; Okuma et al., 1987), gastric contractility

and emptying (Garrick et al., 1987; Maeda-Hagiwara and Tache, 1987),

and gastric serotonin release (Stephens and Tache, 1989). Bilateral

cervical or subdiapliragmatic vagotomy, or atropine, completely reverse all these changes, which indicates that the vast array of peripheral actions of TRH are highly dependent on vagal and cholinergic pathways

(Tache et al,, 1989a). Brain mapping studies revealed that the sites of action of TRH are primarily located at the dorsal vagal complex including the dorsal motor nucleus of the vagus, nucleus tractus solitarius and area postrema, and the nucleus ambiguus (Tache et al,,

1989a), from which the major medullary afferent and efferent vagal fibers are projected to the stomach (Leslie et al., 1982), High concentrations of TRH and its receptors are also detected in the dorsal vagal complex and nucleus ambiguus in immunohistochemical and autoradiographic studies (Tache et al., 1989a). While TRH acts independently of central cholinergic, dopaminergic and serotonergic regulations, the TRH-mediated acid hypersecretion is inhibited by clonidine, an aa-agonist, and the inhibition is reversed by yohimbine, an aa-antagonlst, and by phentolamine, a non-selective antagonist ofai anda 2 receptors (Maeda-Hagiwara et al., 1984; Tache et al,, 1989a), 38

Taken together with the findings that the release of TRH into the

median eminance is increased in rats subjected to cold treatment

(Arancibia et al., 1983), and the fact that the time course for TRH-

induced ulcer formation in the glandular stomach is very similar to that

for cold-restraint stress ulcers, It has been proposed that TRH Js the

central mediator responsible for cold-restraint stress-induced ulcers in

rats (Tahce et al., 1909b). Moreover, TRH antiserum has been shown

to inhibit gastric ulcers produced in rats by cold-restraint (Bagarain et

al., 1984).

1.4.3 Cysteamine-induced duodenal ulcers

In contrast to a myriad of experimental models of gastric ulcers, a

simple and reproducible animal model of duodenal ulcer production was not available until the advent of the use of cysteamine by Selye and

Szabo (1973), Along with cysteamine, other experimental duodenal

ulcerogens such as proplonitrile, a compound structurally related to cysteamine (Szabo et al., 1979), mepirlzole, a non steroidal anti­ inflammatory drug (Okabe et al., 1982) and l-methyl-4-phenyl-l, 2,

3,6-totrahydropyridine (MPTP), a dopaminergic neurotoxin (Szabo et al., 1985), have recently been employed. A single dose of cysteamine given subcutaneously or orally consistently produces solitary duodenal ulcer located on the anterior antimesenterlc wall of the proximal duodenum within 24 hrs in rats, and often perforates into the liver

(Selye and Szabo, 1973; Szabo, 1978). Morphologic and histologic studies revealed that intracellular changes ranging from endoplasmic 39 reticular swelling to mitochondrial disruption occur in the absorptive cell of the proximal duodenal microvilli as early as 30 mins after cysteamine treatment (Pfeiffer et al., 1987). The sequence of events leading to duodenal ulceration by cysteamine starts from intracellular alterations to necrosis and exfoliation of absorptive cells in duodenal microvilli, and followed by microvilli amputation, inflammation of the lamina propria, and finally localized ulcer formation (Poulsen and Szabo,

1977; Szabo, 1987).

The pathogenesis underlying the cysteamine-induced duodenal ulcers in rats is multifactorial. Early studies suggested that a dose- dependent Increase in gastric acid secretion in rats stimulated by cysteamine is the causative factor of duodenal ulcers since vagotomy, anticholinergic agents, antacid and pyloric ligation completely prevented the duodenal ulcerations by cysteamine (Ishli et al., 1976; Robert et al., 1974; Szabo et al., 1979). The enhanced gastric acid secretion has been suggested to be associated with the cysteamine-induced rapid depletions (within 15-30 mlns after cysteamine treatment) of dopamine and somatostatin, two endogenous acid-inhibitory mediators (Szabo et al., 1987; Szabo and Reichlin, 1983), elevation of serum gastrin level

(McIntosh et al., 1984), and delay in gastric emptying (Poulsen etal.,

1982). Dopamine agonists and somatostatin have been shown to prevent cysteamine-induced duodenal ulcers (Gallagher et al., 1987; Schwedes et al., 1977). In contrast, Robert et al. (1974) demonstrated decreases In both the volume and H+ output of gastric acid secretion In pyloric- ligated rats treated with cysteamine, while the concentration of the acid 40

was unchanged.

On the other hand, cysteamine has been shown to weaken the

duodenal mucosal defense mechanisms by inhibiting HCOs- secretion from the duodenal epithelium and the pancreas, predisposing the proximal duodenum to mucosal acid overload, increased luminal Il+ back diffusion Into the mucosal tissue, and ulcer formation (Briden et al.,

1985; Ohe et al., 1982, 1988), Moreover, recent findings propose a role for the Brunner's glands in cysteamine-induced duodenal ulcers in rats since a marked reduction of alkaline mucus secretion from these glands was recorded after cysteamine administration (Kirkegaard et al., 1981;

Koga e t al., 1989). The Brunner's glands have been identified to contain epidermal growth factor (EGF) which acts by inhibiting gastric acid secretion and promoting cell regeneration (Poulsen etal., 1988), and EGF has been shown to inhibit cysteamine-induced duodenal ulcers in rats (Kirkegaard et al,, 1983). In contrast to its inhibition of gastric motility, cysteamine markedly stimulates duodenal motility within 5-10 mins after its administration, resulting in improper mix of acidic gastric contents with the duodenal HCOj” secretion and impaired backward flow of pancreatic and biliary alkaline secretions to the proximal duodenum, which predisposes the duodenal mucosa to injurious actions of acid and pepsin, and to duodenal ulcer formation (Takeuchi et al., 1987; Mangla et al., 1989). 41

1.5 Pharmacology of Antlulcer drugs

Since peptic ulcer disease signifies a breakdown of the dynamic

balance between the luminal aggressive factors and the mucosal

protective mechanisms, pharmacological interventions to suppress the hypersecretion of injurious factors and strengthen the mucosal resistance should restore the imbalance and ameliorate the disease state.

Pharmacological researches on antiulcer therapy have predominantly

focused on the reduction of gastric acid secretion from the parietal cell which is primarily regulated by histamine, acetylcholine and gastrin

(Bertaccini and Corruzzi, 1988; Wolfe and Soli, 1988). The introduction of Ha-receptor blockers into the antlulcer therapy demonstrated the effectiveness of the antisecretory approach to ulcer treatment, which further support the old dictum by Schwartz 'no acid, no ulcer'.

Recently, another class of potent antisecretory agents was developed to block the final pathway of H+ secretion, the H+/K+-ATPase activity, from the parietal cell (Sachs et al., 1988). On the other hand, growing knowledge in mucosal defense mechanisms suggests that promotion of mucosal integrity may contribute to a lower rate of relapse ulceration and possible cure of the disease (Whittle and Garner, 1988).

1.5.1 Antisecretory agents

1.5.1.1 H 2 -receptor antagonists

Hi-receptor antagonism was first described by Black et al. (1972) based upon the observations obtained from in-vivo studies that 42 burimamide (Figure 12), the first Ha-selective antagonist developed, dose-dependently antagonized histamine-induced gastric acid secretion in a competitive and reversible manner, while mepyramlne , a typical Hi- receptor antagonist, was ineffective against this action of histamine.

Other in-vivo studies showed that H 2-receptor blockers also inhibit gastric acid secretion stimulated by pontagastrin infusion or vagal stimulation (Black et al., 1972; Black and Shankley, 1987). These non­ specific inhibitory actions of H2-blockers are not due to direct antagonism of the secretagogues, but rather, it has been hypothesized that histamine acts either by potentiating the secretagogue effects of acetylcholine and gastrin, known as the 'permission hypothesis' (Black and Shankley, 1987), or by being the final-common-chemostimulant of gastric acid secretion, known as the 'transmission hypothesis' (Black and Shankley, 1987). To the contrary, in-vitro studies using isolated gastric glands and parietal cells revealed the receptor specificity of the

Ha-receptor antagonists in that they caused parallel displacement of the dose-response curve to histamine, as measured by the inhibition of [ “ C]-amlnopyrine accumulation, but failed to alter the response to carbachol or gastrin (Soli, 1980; Soli and Berglindh, 1987).

Cimetidine, the first clinically useful H 2-receptor antagonist

(Brimecombe et al., 1975), and other newly developed H 2-recep to r blockers such as ranitidine (Wood, 198G), famotidine (Berlin et al,,

1981) and nizatidine (Lin et al., 198G) (F igure 12), have all been shown to inhibit gastric acid secretion stimulated by histamine, gastrin and chollnomimetics in in-vivo studies, - and to inhibit[1(1 C]-aminopyrine 43

CH.CHiWH.

HISTAMINE

H |C -CM^SCJIjCW.HUOfHCH* ) \ H-C = N NSQ,WM, tF H CIMET1D1NE

H»t*/ c\ HM* f a m o t id in e ^cH.SCT.cn.HHomni, F~ \ OW, CfliN|CH,l. NIZATIDINE •m.r/ V l-U.0***01,01*11110*11 CM, ^^0^ cwo, RANITIDINE

s II c h 2c h 2 c h 2c h 3n h - c - n h - c h 3

Burimamida (S H

c h 2 s c h 2 c h 2 - n h - c - n h - c h 3 N— M a t i a m i d e H 3

Figure 12. Structui*es of the Ha-recoptor blockers. 44 accumulation and adenylate cyclase activity in in-vitro studies (Chebret e t al,, 1981; Lin et al., 1986; Sewing, 1984; Soil and Berglindh, 1987).

In general, Hi-receptor blockers effectively inhibit gastric acid secretion in human subjects by 60-80% (Leth, 1987; Howard et al.,

1985; Robert, 1987) and in isolated human gastric glands (Fagot etal.,

1988; Leth et al., 1987), Suppression of gastric acid secretion by the

Hi-receptor blockers has been shown to be highly correlated to the peptic ulcer healing rate (Wood and Mclsaac, 1988). It has been shown that the maximum ulcer healing rate is 80% after 4 weeks of treatment with Hi-receptor blockers, and approaches 95% after 8 weeks treatment

(Achkar, 1989; Wood and Mclsaac, 1988).

Although Ha antagonists are considered as the drugs of choice for treatment of peptic ulcer (Dobrilla et al., 1989), relapse ulceration following cessation of the treatment with these agents is a frequent clinical observation (Whittla and Garner, 1988). It has been reported that the relapse ulceration rate approximates 80% after 1 year and 100% after 2 years among patients treated with Hi-blockers for 8 weeks

(Achkar, 1989 Goodwin, 1988). The mechanisms underlying the frequent relapse ulceration might be related to the decreased capacity of the healed ulcer crater to produce prostaglandin E2 (PGEs) which is an endogenous mucosal protective substance (Miller, 1983; Pugh et al.,

1989). Moreover, lielicobacter pylori colonization in the mucosal epithelial cells of the healed ulcer crater has been suggested to contribute to the relapse ulceration since elimination of this bacteria by colloidal bismuth subcitrate substantially reduces the relapse rate In 45

patients treated with H 2-blockers (Goodwin, 198B).

Another drawback of H2-receptor blocker treatment is the potential

development of gastric carcinoid. Experimental carcinogencity studies

have shown that long-term (2 years) treatment with potent H2-recep to r

blocking agent, such as loxtidlne and SKF93479, induced gastric tumors

of the enterochromaf fin-like cells (Creutzfeldt, 1988; Hakanson and

Sunder, 1986). It has been hypothesized that achlorhydria induced by

H 2-receptor blockade causes a compensatory increase in serum gastrin

level, and the sustained hypergastrinemla leads to enterochromaffin-like

cell hyperplasia, which precedes the development of gastric carcinoid

(Hakanson and Sunder, 1986). Several lines of evidence showed that

administration of H2-receptor antagonist such as cimetidine, ranitidine

or famotidine markedly Increased serum gastrin level in rats within hours (Decktor et al., 1989; Ohe et al., 1983). In human, gastric

carcinoid development in patients with multiple endocrine syndrome type

I and chronic atrophic gastritis has been shown to be associated with enterochromaffin-like cell hyperplasia subsequent to hyergastrinemia

(Creutzfeldt, 1988; Hakanson and Sunder, 1986). Since H 2-recep tor antagonists have induced hypergastrinemla in humans, they may also produce the similar events of carcinogensis in human as it occurs in rats. (Wolfe and Soli, 1988).

H 2-receptor antagonists have been routinely used as prophylactic agents to prevent stress ulcers and related upper gastrointestinal tract bleeding in patients with severe trauma or serious medical illness 46

(Kleinian et al., 19B8; Wilcox and Spenney, 1988). The use of H 2-

receptor blockers in this group of patients Is associated wtih a

significant Increase In incidence of nosocomial pneumonia and resultant

mortality due to bacterial overgrowth in the stomach (Craven et al.,

1986; Kleinian et al., 1988). Since stress ulcer formation is primarily

due to mucosal ischemia, alternative prophylactic agents such as

sucralfate and calcium antagonists, which act by mechanisms other than

gastric acid inhibition, have been under investigation for their potential

usefulness in stress ulcer treatment (Klelman et al., 1988).

1.5.1.2 Antimuscarinic agents

Before the Introduction of H2-receptor antagonists as antisecretory

agents Into antiulcer therapy, muscarinic antagonists, such as atropine

and other chemically related anticholinergics (Figure 13), were

predominantly used to suppress gastric acid secretion In peptic ulcer

patients. These anticholinergic agents decrease basal gastric acid

secretion by about 40 to 50% and inhibit meal-stimulated gastric acid

secretion by about 30% (Soil, 1989a). However, the usefulness of these agents was limited by the development of side effects such as dryness of the mouth, blurred vision, urinary retention and tachycardia, which are due to their extragastric antimuscarinic activities (Londong, 1982;

Robert, 1987).

Pirenzepine is a recently developed tricyclic compound with anticholinergic activities (Figure 13), It differs from the tricyclic antidepressants by its hydrophilic properties which allow only minimum Atropine Sulpliale

PIRENZEPINE

H 0 N— C

N-CH

TELENZEPINE

Figure 13. Structures of nntimuscnrlnic ngents. 48

amount of the compound to penetrate through the blood-braln barrier,

and central actions are generally absent (Londong, 1982; Mislewicz,

1988). Evidence showed that pirenzepine is a selective muscarinic

antagonist which binds with high affinity to Mi-receptors located in the

autonomic ganglia such as the submucosal and myenteric plexuses In the

stomach, and with low affinity to M2-receptors primarily located on

effector cells and nerve terminals (Bertaccini and Corruzzi, 1988;

Feldman, 1984). Isolated parietal cell studies confirmed that the binding

affinity of, and the inhibition of carbachol-stimulatedf ll,C]-aminopyrine

accumulation by, pirenzepine were about 100-fold less tlian that

produced by atropine (Chan and Soli, 1988; Soli and Bergllndh, 1987),

while in-vivo studies have Bhown that pirenzepine is only 5-6 times less

potent than atropine in inhibiting basal or carbachol-stimulated gastric

acid secretion in rats (Bertaccini and Corruzzi, 1988; Riedel et al.,

1988). These data indicate that pirenzepine exerts its antisecretory

action on muscarinic receptors other than the ones located on the parietal cell. However, clinical studies have shown that pirenzepine

inhibits pentagastrln or sham-feeding stimulated gastric acid secretion

to the same extents as atropine with fewer side effects observed

(Konturek et al., 1980; Londong, 1982). Pirenzepine, given at the recommended oral dose of 50mg, inhibits basal and stimulated gastric acid secretion by less than 60%, which is generally less than the 60-80% inhibition produced by currently available Ha-receptor blockers

(Mislewicz, 1988; Robert, 1987). However, It has been demonstrated that the ulcer healing rate in patients treated with pirenzepine or 49

cimetidine is equivalent (Dobrllla et al., 1989). Recent studies have also

shown that near total suppression of gastric acid secretion can be

achieved by the combination of pirenzepine and clmetldine at doses one-

half of their therapeutic dosages (Mislewicz, 1988; Stockbrugger, 1988).

Another new Mi-selective antimuscarinic agent, telenzepine (Eltze

et al., 19B5), has been demonstrated to be 5-25 times more potent than

pirenzepine in inhibiting gastric acid secretion in animals and in humans

(Chan and Soli, 1988; Londong et al., 1987; Riedel et al., 1988).

However, the Mi-selectivity of this compound could not be demonstrated

by Bertaccini and Corruzzi (1988) and, at a concentration 5 times

higher than Its Ki value for the Mi receptor, significant binding to

"non Mi" receptors occurs (Schudt et al., 1988). Since telenzepine has

a marginal selectivity for muscarinic receptor subtypes (M1/M 2), sim ilar

side effects as produced by atropine may be observed at therapeutic

dosages (Soil, 1989a).

1.5.1.3 Anti gastrin agents

The existence of gastrin receptors on the parietal cell has been confirmed by the observation that the binding of radioactive labelled

gastrin analogue,125I [Leu1 “ ] gastrin, correlated positively with a highly enriched isolated cannine parietal cell fraction (Soil et al., 1984; Soli and Berglindh, 1987). Gastrin binding and the subsequent stimulation of parietal cell function, as measured by the accumulation of[li,C]- amlnopyrine, were competitively inhibited by praglumide (Soil et al.,

1984) (Figure 14). Being a derivative of glutaramic acid, proglumide is 50

0 1 H | >CH2—CH2—CH3 HOC—CH2- :h2—c—c—n "'v CH2—CHj—CH3 NH I c—o

Proglumide

o H |

NH G c r I H 9 " °

Cl Benzotript

Figure 14. Structures of antigastrin agents.

4 51

a specific inhibitor of gastrin receptors on the parietal cell and of

cholecystokinin receptors in the pancreatic acinar cell (Hahne et al.,

1981; Robert, 1987). Both in-vlvo and in-vitro studies have

demonstrated that proglumide Inhibited gastric acid secretion by gastrin

(competitive inhibition) or acetylcholine (non-competitive inhibition), but

was ineffective against histamine (Magous and Bali, 1983; Robert, 1987;

Rovati, 1976). Clinical trials revealed that proglumide, given at a

therapeutic dose of lOOOmg/day, inhibited acid secretion by 42% and

produced an ulcer healing rate of 80% after 4 weeks (Rovati, 1976).

However, it is thought that the inhibitory action of proglumide is not

specific and not sufficiently potent to be of clinical usefulness as

compared to the currently available antisecretory agents (Wolfe and

Soil, 1988). Benzotript (Figure 14), a derivative of tryptophan, has

similar actions as proglumide with increased potency against gastric acid

secretion (Magous and Bali, 1983; Hahne et al., 1981).

1.5.1.4 Hf £ K * -ATPase inhibitors

In 1973, Ganser and Forte (1973) identified the gastric

H+/K*-ATPase which was subsequently found to be located on the

smooth membranes of the tubulovesicles and the secretory canalicull of the parietal cell (Smolka et al., 1983). This gastric enzyme Is

stimulated by luminal K* (Wallmark et al,, 1980) and driven by the energy generated from the hydrolysis of ATP. The H+/K+-ATPase is a countertransport pump, electroneutrally exchanging cytosolic H* for luminal K+ (Sachs, 1987; Wallmark et al., 1980), The primary amino acid 52

sequence of this enzyme has been deduced by cloning techniques and

the enzyme is characterized by having a large number of cysteine residues located in the hydrophobic region, as well as one cysteine residue extended into its luminal sector (Sachs et al., 1988; Shull and

Lingrel, 1986). The gastric H*/K*-ATPase represents a unique target for pharmacological intervention, since inhibition of the enzyme activity can totally abolish HC1 secretion from the parietal cell.

Omeprazole, a substituted benzlmidazole (Figure 15), is the first clinically useful inhibitor of the H+/K*-ATPase (Lindberg et al., 1986,

1987). Two other investigational compounds belonging to this class of inhibitors are plcoprazole and timoprazole (Sewing et al,, 1983). It has been shown that omeprazole was effective in inhibiting HC1 secretion in various species, including rat, dog, and human, regardless of whether secretion was stimulated by histamine, carbachol, or pentagastrin

(Larsson et al., 1983; Lind et al., 1983). In isolated gastric glands or purified parietal cells, omeprazole effectively inhibited histamine, db- cAMP, or K+-stimulated gastric acid secretion, as measured by the accumulation of [ lhC]-aminopyrine (Lindberg et al., 1987; Wallmark et al., 1985). Omeprazole Is a permeable weak base (pKa=4) and can be easily charged and trapped within the acid compartments of the parietal cell (Wallmark et al., 1985). The mechanism of action of omeprazole involves acid-induced transformation into an active intermediate, sulphenamide (Lindberg et al., 1987; Wallmark, 1986), which rapidly

Interacts with the luminal cysteine residue of the H+/K+-ATPase to form a dlsulphide Inhibitory complex resulting in HC1 secretion inhibition (Lindberg et al,, 1987; Lorentzon et al., 1985) (Figure 1G). From the autoradiographic studies in mouse, [ 3H]-omeprazole was found to selectively label the catalytic subunit of H*/K*-ATPase located on the smooth membranes of the tubuloveslcles and the secretory canallculi of the parietal cell (Sachs et al., 1988; Helander et al., 1985).

Furthermore, a strong correlation has been demonstrated between the inhibition of acid secretion, H+/K+-ATPase activity, and phosphoenzyme formation in rats treated with omeprazole (Wallmark et al., 1985;

Lorentzon et al., 1985). The inhibitory action of omeprazole was found to be pH-dependent and reversed by the addition of sulfhydryl(-SH) agents such as 3-mercaptoenthanol and dithiothreltol (Im et al.,

1985a,b; Lorentzon et al., 1987).

Omeprazole is the most powerful and long-acting inhibitor of acid secretion available for use in humans. Clinical studies have demonstrated that omeprazole, given as a single dose of 40 mg, significantly inhibited acid secretion for 3 days (Misiowicz, 1988), and treatment with 20 or 30 mg omeprazole daily after 1 week decreased

24-hr intragastric acidity by 90% and 97%, respectively (Soli, 1989a;

Mislewicz, 1988). When given at 30 mg daily, omeprazole produced an ulcer healing rate of 75% after 2 weeks and 98% after 4 weeks, which is more effective than that achieved by Ha-receptor blockers (Farup et al., 1989 Dobrilla et al., 1989). However, follow-up studies do not disclose any difference between the incidence of relapse ulceration after treatment with omeprazole, or H2-receptor antagonists (Misiewicz, 1988).

One study showed that the overall relapse rate after 6 months upon 54

‘ Timoprazole

H 0 CH3 HaC > s - c h 3< P > Picoprazote H3COC 0

H 0 CH, OCH, Omeprazole H3C0

S C H 3 2 0 5 1

CH,

CH,

S C H 2 8 0 0 0

Figure 15. Structures of the H*/K*-ATPase inhibitors. («) Secretory canaliculus

(*>) Secretory canaliculus

-A'

Cytosol

Blood

(C) Secretory canaliculus -Al

Cytosol

Blood

Figure 1G. Events lending to inhibition of gastric acid secretion by omeprazole analogue, tlmoprozole, within the parietal cell, (a) Accumulation of protonated timoprazole in the acidic compartment; (b) transformation of timoprazole to sulplienamide; and (c) the structure of the enzyme-lnhibltor complex. (Adapted from Lindberg et ni., 1987). 56 cessation of 2-week treatment was G2% (Farup et al., 1989). Another drawback of omeprazole treatment, which is common to all potent antisecretory agents, is the development of gastric carcinoid due to prolonged hypergastrinemla subsequent to sustained achlorhydrla

(Creutzfeldt, 1988; Decktor et al., 1989). Carcinogenicity studies have demonstrated that treatment with high dose (140 mg/kg) omeprazole daily for 2 years in female rats produced gastric carcinoid of the enterochromaffin-like (ECL) cell in 40% of the rats (Havu, 1986). ECL cells hyperplasia has also been shown to occur during short-term (10 weeks) treatment in rats (Larsson et al., 1986). It is, therefore, important to define the safety of the drug in long-term treatmnet of diseases such as Zollinger-EUison syndromes.

In addition, SCH28080 and SCH32651 (Figure 15), and calcium antagonists have been shown to inhibit gastric acid secretion by blocking the H*/K*-ATPase activity in isolated gastric glands and In mucosal membrane vesicles (Scott et al., 1987; Sewing and Hannemann,

1983; Im e t al., 1984). SCH28080 and SCH32G51 are protonable amines with similar inhibitory profiles as those of omeprazole (Sachs et al.,

1988; Scott et al., 1987); however, the inhibition of the enzyme by these compounds was not reversible upon addition of 5-mercaptoethanol or dithiothreitol, but rather, was readily reversible by increasing the concentration of KC1 used to stimulate the enzyme. These data suggest that SCH28080 and SCH32G51 reversibly and competitively inhibit the activation of the K* site of the H*/K+-ATPase (Sachs et al., 1988; Scott e t al., 1987). On the other hand, in the case of the inhibitory action of 57 calcium antagonists, such as verapamil, gallopamil, and TMB-8, on HC1 secretion, it is not so conclusive that inhibition of acid secretion might be due to the interaction with the high-affinity K+ site of the gastric

HVK*-ATPase (Sewing and Hannemann, 1983; Im et al., 1984) or dependent on a non-specific accumulation of the antagonists in the acidic compartments of the parietal cells (Herling and Ljungstrom,

1988).

1.5.1.5 A ntacids

Aluminum hydroxide [Al(OH) i ] is the most commonly used cationic antacid, which is clinically more effective in healing peptic ulcers than the anionic antacids (e.g., calcium carbonate) (Fordtran et al., 1973;

Malagelada and Carlson, 1982). It is generally believed that the antiulcer action of antacids is predominantly associated with their gastric acid buffering capacity. However, recent findings have revealed that, Instead of providing antacid with 800-1000 mEq neutralizing capacity daily to promote ulcer healing (Ippoliti, 1982; Peterson et al.,

1977), peptic ulcers can be healed by antacid with buffering capacity as low as 100-120 mEq daily (Berstad and Weberg, 198G; Blum, 1985), and the healing efficacy is comparable to that of clmetidine (Blum, 1985).

This raises the question as to whether antacid accelerates ulcer healing by mechanisms other than acid neutralization, such as binding of bile acids and Inactivation of pBpsin (Soli, 1989a; Weberg et al,, 1985), since 120 mEq neutralizing capacity produces modest acid buffering

(Soli, 1989a; Weberg et al., 1985) and interaction with food further 58 reduces In-vivo antacid neutralizing power (Berchtold et al., 1985;

Halter, 1988).

Several lines of evidence have demonstrated that Al(OH)i- containing antacids induce luminal release of prostaglandin 2 E (PGE 2), an endogenous cytoprotective substance (see section 1.5.2.1). Such an effect cannot be induced by AHOj, AICI3 , CaCOs, or Mg(OH)2 (H alter,

1988; Lanz et al., 1985; Szelenyi and Postius, 1985). Since sucralfate

(see 1.5.2.2), an effective antiulcer agent with no effect on intragastric pH, also contains aluminum salt and releases PGE2 into the gastric juice

(Szelenyi and Postius, 1985), it would be expected that the ulceroprotective mechanism of Al(OH) 3 -containing antacids is mediated b y PGE2 . Recent studies confirmed this alternative action of Al(OH)3- containing antacids, in that A1(0H)3, Mylanta II, and Trigastrll significantly reduced absolute ethanol-induced deep mucosal necrosis in rats and aspirin-induced gastric microbleeding in man, which highly resemble the mucosal protective action of PGs (Domschke et al., 1986;

Hollander et al., 1986; Tarnawski et al., 1985). Therefore, Al(OH)3- contalning antacids may possess dual antlulcer actions - acid neutralizing activity and gastric mucosal protection.

1.5.2 Mucosal protective agents

1.5.2.1 Prostaglandins

In addition to their well-known antisecretory actions against basal and secretagogue-stimulated gastric acid secretion in animals and in man 5 9

(Robert, 1987; Wolfe and Soli, 1988; see section 1.3), prostaglandins

(PGs) of the E series (PGEi and PGE2) have been demonstrated to prevent gastric mucosal necrosis In rats Induced by noxious agents such as absolute alcohol, concentrated HC1 and NaOH, and boiling water in a dose-dependent manner at doses that have no effect on gastric acid secretion (Robert et al, 1979). The gastric mucosal protective effect of

PGs against necrotizing agents independent of acid inhibition is termed as 'cytoprotection' (Robert et al., 1979; Szabo, 1988). However, the idea of cytoprotection by PGs, which is largely derived from observations of a marked reduction or absence of macroscopically visible hemorrhagic mucosal lesions induced by necrotizing substances, is inconsistent with the findings from histoglcal and microscopic studies that PGE2 does not protect gastric mucosal epithelial damage produced by absolute alcohol, but reduces the depth of mucosal damage extending to the gastric glands (Lacy and Ito, 1981; Miller, 1983). This discrepancy is also reported by Wallace et al. (1982) that the reduction of alcohol-induced gastric lesion area by PGE2 is directly related to reduction of deep mucosal vasocongestion and not to any enhanced resistance of the luminal epithelium to the damaging effect of alcohol, as measured by changes in transmucosal potential difference and net fluxes of Na+ and H* (Szabo, 1988). Therefore, instead of using the term

'cytoprotection' to describe the actions of PGs, it should be modified to

'mucosal protection1. The mechanisms underlying the mucosal protective action of PGs include prevention of gastric mucosal barrier disruption, stimulation of mucus and bicarbonate secretions, enhancement of gastric 60

mucosal blood flow, maintenance of gastric mucosal sulfhydryl (-SH)

compound, and promotion of cell regeneration (Graham et al., 1988;

Miller, 1983; Szabo, 1988).

Since naturally occurring PGs are unstable and short-acting,

several analogues of PGEi (misoprostol and rioprostil) and PGE2

(arbaprostil, enprostil and trimoprostil) have been synthesized that are

more potent than their natural counterparts, but that also resist rapid

metabolism, making them suitable for oral administration (Dobrilla etal.,

1989; Hawkey and Walt, 1986; Mlsiewlcz, 1988). Misoprostol Is the first

'cytoprotective' agent approved for clinical use for the prevention of

gastric ulcers in high-risk patients taking non-steroidal anti­ inflammatory drugs (NSAIDs) (Graham et al., 1988). Binding studies demonstrated that [ 3H]-misoprostol stereospecifically binds to the E-type

PG receptor with high affinity in the canine enriched parietal cells

(T sai et al., 1987). Interaction of the receptor with misoprostol dose- dependently (50-200 ug) inhibits basal, histamine- and meal-stimulated gastric acid secretion in man for up to 3 hours (Misiewicz, 1988;

Wilson, 1987). In addition, misoprostol exhibits Its mucosal protective actions by increasing gastric mucosal blood flow in dogs at non- antisecretory doses (Gana et al., 1989); whereas, increase in mucosal blood volume in humans can only be achieved at doses possessing antisecretory action (Sato et al., 1987). Moreover, it has been demonstrated that misoprostol stimulates HCOi- and mucus secretion in animals and in man (Gana et al., 1989; Nicholson, 1988). Evidence generated from animals and humans studies shows that misoprostol 61 protects the gastric mucosal damage caused by NSAIDs and ethanol In a dose-depdendent manner over a range from 25 to 200 ug, which Is significantly superior than clmetidine and sucralfate (Bauer et al.,

1986; Soli, 1989a). However, this range also produces antisecretory actions of misoprostol, and it Is difficult to differentiate the mucosal protective effect from the antisecretory effect.

Ulcer healing efficacy of misoprostol Is dose-dependent in that at low doses (25-50 ug four times daily), which have minimum effect on acid secretion, were no better than placebo; while high doses (100-200 ug four times daily), which possess comparable antisecretory action as cimetidine, were as effective as cimetidine (Achkar, 1989; Hawkey and

Walt, 1986). Therefore, it is believed that although misoprostol is referred to as a cytoprotective agent, it is not effective in the treatment of peptic ulcers unless it is prescribed at a high enough dosage to interfere with acid secretion. This implies that its cytoprotective properties, which are so dramatic in aucte animal experiments, play a smaller part in ulcer healing In humans (Hawkey and Walt, 1986; Achkar, 1989), The most common side effect associated with misoprostol treatment is diarrhoea which occurs in 15% of the patients. A second drawback of this agent is the potential abortifacient activity (Achkar, 1989).

1.5.2.2 Sucralfate

Sucralfate (Figure 17), a sulfated disaccharide complex with aluminum hydroxide, is a locally-acting antiulcer agent with no effect on G2

CHjOR

ROCH

OR RO O R? / CHjOR

H OR OR H R = S O JAI,(OH)1

Figure 17. Structure of sucralfate. 63

gastric acid secretion. At pH below 3.5, sucralfate becomes a condensed

adhesive substance preferentially coating the eroded mucosal surface

(Soli, 1989a). It has been suggested that, under acidic environment,

some parts of aluminum hydroxide moieties are dissociated from sulfate

anions of sucralfate molecules. The sucralfate polyanions electrostatically

bind the positively charged proteinacious substrates at the ulcer site to

form a gel layer (Koba, 1988; Spiro, 1982), which acts as a protective

barrier against H* back diffusion, hydrolytic action of pepsin, and

mucosal damaging action of bile acids (Garnett, 1982; Spiro, 1982;

Tranawski et al., 1987), Moreover, in-vitro studies also showed that

sucralfate deactivates pepsin and adsorbs pepsin and bile acids

(Tranawski et al., 1987). Although sucralfate does not alter Intragastric

pH, it Is believed that local acld-neutrallzation beneath the protective

barrier does occur (Garnett, 1982). In animal studies, sucralfate has been shown to protect the gastric mucosa against acute ulcerations induced by NSAIDs, restraint stress, and absolute ethanol (Okabe et al., 1983; Tranawski et al., 1987). This gastroprotective action has also been confirmed in humans in that pretreatment with sucralfate

significantly reduced aspirin-induced gastric bleeding (Konturek etal.,

1987), The antiulcer mechanism of sucralfate has been shown to be associated with the increased biosynthesis and luminal release of PGEa

(Konturek et al., 1987; Tranawski et al., 1987). Sucralfate has also been demonstrated to stimulate amphibian gastroduodenal HCOj- secretion directly by the aluminum component of sucralfate and indirectly by the release of PGE2 (Crampton et al., 1987; 1988). Clinical trials demonstrated that the healing rate after 4 weeks of

sucralfate therapy ranges from 60% to 83% in duodenal ulcer patients,

and From 47% to 62% in gastric ulcer patients, which is comparable to

that of cimetidine (Marks, 1987). Maintenance therapy with sucralfate

has also been shown to prevent ulcer relapse in cimetidine-treated

patients (Marks, 1987; Takemoto et al., 1987). Sucralfate has proven to

be effective for the prevention of stress-induced gastrointestinal

bleeding with an advantage over the conventional prophylactic therapy

u sin g H2-antagonists or antacids in that there is a substantial reduction

(50%) in the incidence of nosocomial pneumonia in ventilated patients

treated with sucralfate as compared to those treated with antacids or

H 2-receptor blockers (Tryba and Mantey-Stiers, 1987), Recent findings

showed that sucralfate has antibacterial action against E. coli and P. aeruginosa (Tryba and Mantey-Stiers, 1987).

1.5.2.3 Colloidal bismuth subcitrate

Colloidal bismuth subcitrate (CBS) is a complex bismuth salt of citric acid and is the active ingredient in De-Nol® . The chemical formula of CBS is(Bix(0H)y(CeHs07)z] . The citrate moiety (CsHsO?) together with the trivalent Bi(OH)i can generate many possible salt formations (Hall, 1989; Simjee, 1988). At neutral or alkali pH, CBS is a colloid, whereas it begins to precipitate to form bismuth oxychloride and bismuth citrate under acidic environment at pH 3.5-4 (Hall, 1989;

L ee, 1982). It has been demonstrated that CBS preferentially coats the crater of rat gastric ulcers (Hall, 1989), which has been confirmed In 6 5 ulcer patients in that 90% of the bismuth can be recovered from the ulcer site after a single oral administration (Lee, 1982), In-vltro studies also revealed that CBS binds to mucus to form a complex that significantly retards H+ diffusion (Lee, 1902), which leads to the proposal that CBS protects gastric mucosal insults induced by ethanol, acidified aspirin, and restraint stress, and enhances healing of gastric and duodenal ulcers via the formation of the occlusive complex around the ulcer crater to prevent H* back diffusion and digestive actions of pepsin and bile acids (Konturek et al., 1987, Lee, 1982). In addition, recent findings showed that CBS forms pH-dependent complexes with epidermal growth factor (EGF) in vitro and accumulates EGF In the ulcer crater in rats to accelerate the re-eplthelialisation and tissue repair of the ulcerated mucosa (Konturek et al., 1988). Furthermore,

CBS has been shown to stimulate the luminal release of PGE2 and the subsequent gastroduodenal HCOj- secretion in a dose-dependent manner in dogs and in man, which can be blocked by indomethacin (Konturek et al., 1987a,b). CBS was about four times more potent than sucralfate at stimulating PGEz synthesis (Hall, 1989). The overall healing efficacy of CBS in gastroduodenal ulcers is 75% after 4 weeks of treatment, which is as effective as that of cimetidine and ranitidine (Tytgat, 1987;

Simjee, 1988). One significant advantage of CBS over the H 2- antagonists is substantially lower relapse ulceration rate after CBS healing than cimetidine or ranitidine healing (Simjee, 1988; Tytgat,

19B7). The lower relapse rate after CBS healing has been attributed to its antibacterial action against helicobactor pylori which has been 66

suggested to be a causative factor of chronic gastritis and subsequent

gastroduodenal ulcer formation (Marshall et al., 1987; Miller, 1989).

1.6 Calcium Antagonists and Peptic Ulcer Disease

As mentioned above, various gastroduodenal functions are ultimately dependent on the calcium Ion as their signal transducer. The release of histamine from the mast cell, acetylcholine from the nerve terminal, and gastrin from the G-cell are all calcium-depdendent processes (Chakravarty, 1986; Hertz and Cloarec, 1989; Wolfe and Soli,

1988). In addition, binding of these secretagogues to their respective receptors located on the basolateral membrane of the parietal cell stimulates transmembraneous calcium influx and release of calcium from intracellular stores resulting in a rise of cytosolic calcium which acts as a second messenger to regulate H* secretion by the H*/K+-ATPase pump at the apical membrane of the cell (Chew, 1986; Chew and Brown, 1986;

Negulescu and Machen, 1988). It has also been demonstrated that infusion of calcium gluconate stimulates gastric acid secretion in humans

(Kirkegaard et al., 1982; Sonnerberg et al., 1984). M oreover, excitatlon-contraction coupling of the gastric muscle wall and the gastric vascular smooth muscle is also dependent on the presence of extracellular and intracellular calcium (Spedding, 1988; Wolfe and Soli,

1988). Since the pathogenesis of peptic ulcer disease is suggested to be associated with the abnormalities of these gastric functions, it prompted investigators to study the role of calcium antagonists in the treatment of peptic ulcer disease. Based on their cellular site of action, the 67

calcium antagonists are broadly classified into two groups: the calcium

channel blockers and the intracellular calcium antagonists (Rahwan,

1983; 1989).

1.6.1 Calcium channel blockers

Calcium channel blockers can be subdivided into three major classes consisting of the 1,4-dihydropyridines (e.g., nifedipine, nicardipine, nitrendipine, and nimodipine), the phenylalkylamines (e.g., verapamil, gallopamil, and tiapamil), and the benzothiazepines (e.g., dlltiazem) (Triggle and Janis, 1987) (Figure18). Some members of the calcium channel blockers, including nifedipine, verapamil, and dlltiazem, are well-established therapeutic agents employed in cardiovascular diseases such as angina, hypertension, and supraventricular tachycardia, due to their vasodilatory action and atrioventricular conduction depressant activity resulting from blockade of the slow inward calcium channel (Triggle and Janis, 1987; Nayler, 1988).

Potential therapeutic actions of calcium channel blockers on disorders other than cardiovascular diseases have also been examined, and include achalasia (Castell, 1985), gastrointestinal motility disorders (Defeudis and Christen, 1989; Speddlng, 1988), peptic ulcer disease (Glavin,

1989; Speddlng, 1988), and epilespsy (Binnie, 1988; Meyer et al.,

1986). The chemical heterogeneity of these calcium channel blockers suggests different sites and mechanisms of actions, which are confirmed by radioligand-binding studies that demonstrate three discrete, alios ter Ically linked binding sites for the three major classes of calcium GO

n i CN i Me0' O - 9 ‘CH*J3f/CHiCHi~ Q “ 0Me OMe MeO pr‘ M* OMe Me 'OM e R* H, Verapamil R -O M e, DGOO/Galiopomil Tiapomil

a,-NOz MeOOC■ vykyCOOM ' A ' ' e EtOOC COOMe Me N Me Me n H CHiCH^Mei Nifedipine Felodipine Diltiozem

NO:

MeOOC COOCKyCHiNMeCHjPh MeOOC COOEt MeOOC COOCHjCHM*!

M e^'fJ ''Me

Nicardipine Nitrendipine Nisoldipirve

-N O j

Me^HOOC-v-AyCOOCt^CHjOMe MeOOC COOCHMei

Me [{ ''M e Me H Me Nimodipine PN 2 0 0 ttO

Figure 10. Struclu ros of tliG tiiroc innjor classes of cnlcium chnnnel bJcokoi’S. 69

channel blockers (Godfralnd et al., 1906; Hurwltz, 1986; Triggle and

Janis, 1987). Among the various calcium channels identified, which

include voltage-operated calcium channels, receptor-operated calcium

channels (Dolton, 1979; Meisheri et al., 1981), calcium-leak channels

(Loutzenhior et al., 1985) and stretch-activated calcium channels

(Nayler, 1980; Van Brecman et al., 1986), the vbltage-operated calcium channel is the primary site of action of the calcium channel blockers, and, at higher doses, receptor-operated calcium channels are also susceptible to blockade by the calcium channel blockers (Vanhoutte and

Paoletti, 1987; Godfraind et al., 1986). Recently, It has become apparent that there are three subtypes of voltage-operated calcium channels (Godfralnd et al., 1906; Nowycky et al., 1905; Scott and

Dolphin, 1989), The most common subtype, identified in many cells, Including the cardiac and vascular smooth muscle cells, is the L-type calcium channel which carries large conductance providing long-lasting current at strong depolarization. A second type, also found in sinoatrial pacemaker cells, termed the T-type cnlcium channel, Is activated at much more negative membrane potentials than the L-type and carries a small transient current. In dorsal root ganglion, N-type calcium channels are identified which are activated at strong depolarization and inactivated at strongly negative potential (Nayler,

1988; Scott and Dolphin, 1989). It has been suggested that different types of channels exert different cellular Functions. For example, T- typo calcium currents influence sinoatrial pacemaker activity in the heart, whereas L-type channels contribute to maintain cardiac 70

contractility, and N-types participate in neurotransmitter release

(Reuter and Porzig, 1988; Nayler, 1988). The L-type calcium channel is

susceptible to all three major calcium channel blockers, while the T-type

channel is only suppressed by verapamil, and the N-type channel is

insensitive to all calcium channel blockers (Defeudis and Christen, 1989;

Tang et al., 1988; Nayler, 1988; Yaari et al., 1987).

While nifedipine exhibits preferential effects on vascular smooth

muscle, verapamil and dlltiazem express equiactive effects on calcium

channels in cardiac and vascular smooth muscle, which helps to explain

their profound depression on the electrophysiology and contractility of

the cardiac muscle (Henry, 1980; Hurwitz, 1986). In addition, verapamil

and dlltiazem, but not nifedipine, exhibit use-dependent blockade of the

calcium channel, in that their inhibitory activities increase with

increasing frequency of stimulation of the channel. This Indicates that verapamil and dlltiazem demonstrate little or no affinity for the calcium channel in the deactivated state, but interact to a significant degree with activated or inactivated ion channels (Hurwitz, 1986; Triggle and

Janis, 1987). This biophysical property of verapamil and dlltiazem, together with evidence of a high density of T-type calcium channels identified in the rabbit sinoatrial pacemaker cells, provide further understanding of their antiarrhythmic activity which is likely, at least for verapamil, mediated by blockade of T-type calcium channel (Tang et al., 1988, Bean, 1989; Yaari et al., 1987). 71

1.6.2 Intracellular Calcium Antagonists

Instead of blocking different types of membrane calcium channels, intracellular calcium antagonists bypass the complexities of the membrane channels and exert their pharmacological actions by blocking intracellular calcium receptors (e.g., troponin or calmodulin), preventing calcium mobilization from the intracellular stores, facilitating calcium sequestration by Intracellular organelles, enhancing calcium efflux from the cell, or interfering with the contractile proteins

(Rahwan, 1903; 1989). This group of calcium antagonists consists of methylenedioxyindenes (MDIs), magnesium, sodium nitroprusside, diazoxide, and the trimethoxybenzoate compounds (TMB-8) (Rahwan,

1983; 1989) (Figure 19).

This section will only focus on the pharmacology of the MDIs which were developed by Witiak et al. (1974) and were later found to possess calcium antagonistic action (Rahwan et al., 1977). The prototypes of the tertiary amine MDI series are 2-n-propyl-3-dimethylamino-5,

G-methylenedioxyindene HC1 (pr-MDI) and 2-n-butyl-3-dimethylamino-5,

6-methylenedioxyindene HC1 (bu-MDI) (Figure 19). Structure-nctivity studies revealed that propyl- or butyl-substituent at the 2 position, the

5,6-methylenedioxy bridge, and the tertiary aminoindene ring are essential for intracellular calcium antagonistic activity (Witiak et al.,

1982; Rahwan, 1989).

In the heart, In vitro studies using isolated guinea pig atrium demonstrated that pr-MDI exhibit intrinsic negative inotropic effects 72

.0- < CHj o- • N - " Cl CH^ CHj CH^ ^CHj PR-MDI BU-MDI

CH,0. Q b ^ \ n p facH, ch ,o^ O > -c “° - ,ci V b“ N( y— ' CHaCHs CHJO

TMB-8

0 2 ‘ SV

N*“ c ' S * A SODIUM NITROPRUSSIDE

N ^ C H ,

0 0

DIAZOXIDE

Figure 19. Structures oT some Intracellular calcium antagonists. 73 without blocking the slow inward calcium channels nor other presumptive membrane routes of calcium entry into the myocardial cells

(Lynch and Rahwan, 1982), and this effect correlates well with the accumulation of 1

(Rhawan, 1989). In addition to its calcium antagonistic action, It has been demonstrated that pr-MDI inhibited histamine-activated (H 2 - mediated) guinea pig ventricular adenylate cyclase activity with a pA2 of 6.46 as compared to a pA2 of 6,10 for the specific II2 -recep to r antagonist cimetidine (Johnson, 1979; Johnson and Grupp, 1979).

Blockade or the H2 -receptor coupled adenylate cyclase activity by pr-

MDI does not appear to be directly related to its calcium antagonistic action nor to the inhibition of positive inotropic action of histamine

(Rahwan, 1989). 74

In vascular smooth muscle, pr-MDI has been shown to inhibit the

contractile effect of U44069[a stable analogue of prostaglandin 2 H which

contracts vascular smooth muscle by mobilizing intracellular calcium] on the isolated rat aorta in a calcium-free medium, confirming an

intracellular site of action of the MDI (Heaslip and Rahwan, 1982). The

tertiary MDIs also inhibited the two intracellular calcium-depdendent

phases of norepinephrine-induced aortic contraction in the absence of

extracellular calcium, while nifedipine had no effect on either response

(Heaslip and Rahwan, 1983). Moreover, the MDIs Inhibited the

intracellular calcium-dependent constriction of human umbilical veins

induced by barium chloride and U46G19 (Mak, 1984). Direct evidence of

the intracellular calcium antagonistic action of pr-MDI was obtained from

in-vitro studies using chemically-skinned rat caudal arterial strips that

contraction of the skinned artery with free calcium solution was

significantly obtunded by trifluoperazine (a calmodulin antagonist) but not by pr-MDI, while the contraction of the skinned artery evoked by high concentration of caffeine (which stimulates release of calcium from the sarcoplasmic reticulum) in the absence of extracellular calcium was significantly obtunded by pr-MDI but not by nifedipine. It was concluded that pr-MDI acts intracellularly to block calcium mobilization from the sarcoplasmic reticulum without directly interfering with the regulatory proteins (Wong and Rahwan, 1988). Biochemical studies also confirm that the MDIs do not act as calmodulin antagonists (Weishaar et al., 1983). 75

The MDIs were found to block the spasmogenic action on estrogen-

treated rat uterus of PGE2 , oxytocin, barium, and acetylcholine

(Rahwan et al., 1977). Moreover, they also blocked the contractile

effect of histamine on the Isolated guinea pig Ileum, and of acetylcholine

on the isolated rat ileum (Rahwan et al., 1977). In a stimulus-secretion

coupling model using the perfused bovine adrenal medulla, the MDIs

were also found to block carbachol-induced catecholamine secretion from

chromaffin cells by interfering with the action of intracellular calcium

(Plascik et al., 1978). The sensitivity of stimulus-secretion coupling

mechanisms In vitro to the inhibitory effects of the MDIs was found to

be 500-1000 times greater than the sensitivity of excitation-con traction

coupling mechanisms (Piascik et al., 1978). It has also been

demonstrated that the MDI inhibits caffeine-induced contractures (which

are mediated by intracellular calcium) of the isolated rat hemidiaphragm

skeletal muscle both In the presence and in the absence of extracellular

calcium (Rahwan and Gerald, 1981), and calcium mobilization from the

sarcoplasmic reticulum upon stimulation of skeletal muscle as evidenced

by the reduction of activation heat (Burchfield et al., 1982).

1.6.3 Effects of calcium antagonists on experimental ulcers

It has been unequivocally demonstrated that cold-restraint stress- induced gastric ulcers In rats can be prevented by pretreatment with calcium channel blockers such as nifedipine, nitrendipine, verapamil, and dlltiazem (Glavin, 1988; 1989; Ogle et al., 1985; Walt et al., 1985).

However, inconsistent results are observed In ellianol-induced gastric 76 ulcer studies. While verapamil was shown to worsen or have no effect on this type of experimental ulceration in rats (Glavin, 1988; 1989;

Hertz and Cloarec, 1989), Ghanayem et al. (1987) demonstrated significant protection by verapamil and diltiazem against ethanol-induced gastric ulcers. Moreover, the lntter group of investigators (Ghanayem et al., 1987) demonstrated ulceroprotective action of verapamil and diltiazem against indomethacln-induced gastric ulcers in rats, whereas

Hertz and Cloarec (1989) did not reveal any antiulcer action of verapamil against this type of experimental ulcers. On the other hand, basal gastric acid secretion is significantly inhibited by the calcium channel blockers including nifedipine, verapamil and diltiazem, but not by flunarizine and cinnarizine (Glavin, 1988; 1989; Hertz and Cloarec,

1989; Brage et al., 1986). In addition, differential actions against different secretagogues among the calcium channel blockers have been observed in that verapamil, diltiazem and cinnarizine inhibited pentagastrin-induced acid secretion in rats, whereas nifedipine only inhibited liistamlne-induced gastric acid secretion (Bouclier and

Speddlng, 1985) .

Studies in Isolated rabbit gastric glands demonstrated that verapamil, but not nifedipine, caused a concentration-dependent inhibition of [ lftCJ -arninopyrine accumulation and oxygen consumption during carbachol, histamine, and db-cAMP stimulation (Herling and

Ljungstrome, 1988; Im et al., 1984). In addition, experiments with isolated guinea pig parietal cells further revealed that verapnmil and gallopamil inhibited gastric acid secretion stimulated by histamine, db- 77

cAMP, and KC1 in a dose-dependent manner (Sewing, 1984; Sewing

Hennemann, 1983) and, in isolated hog membrane enriched with

(H*+K*)-ATPase, verapamil competitively inhibited K+-stlmulated ATPase

activity (Im et al,, 1984), which lead to the conclusion that the

antisecretory action of verapamil and gallopamil in-vitro is mediated not

by channel blockade, but by interference of the H*/K+-ATPase activity

(Sewing and Hennemann, 1983; Im et al., 1984). However, other in-

vitro studies showed that verapamil was ineffective against acid

secretion Induced by histamine, carbachol, theophylline, and db-cAMP

(Erjavec and Stanovnik, 1989; Kirkegaard et al., 1982). Since the

concentration of calcium antagonists needed to depress acid secretion in

many instances are rather high, in the range exhibiting inhibitory

effects on cardiac and vascular muscle, the specificity of their effect is questionable (Erjavec and Stanovnik, 1989; Hertz and Cloarec, 1988).

Herling and Ljungstrom (1988) suggested that inhibition of acid production in vitro by verapamil is due to a non-specific accumulation of the drug in the acidic compartments of the parietal cell, leading to an impaired function of the II*/K*-ATPase pump.

Clinical studies also revealed equivocal results. While verapamil and nifedipine have been shown to significantly inhibit both basal and pentagastrin-stimulated gastric acid secretion in human volunteers

(Caldara et al., 1985; Kirkegaard et al., 1982; Sonnerberg et al,,

1984), other studies demonstrated no antisecretory action of verapamil against basal, histamine, pentagastrin, or modified sham-feeding- stimulated gastric acid secretion In healthy human subjects (Aadland 78 and Berstad, 1983; Levin et al., 1983).

The conflicting results reported by different laboratories might stem from the fact that different experimental models were used to study the antiulcer and antisecretory actions of the same calcium channel blockers Also, from the viewpoint of the diversity and complexity of membrane calcium channel, there are uncertainties surrounding the existence, function, type, or state of voltage-depedent calcium channels in the parietal cell and the gastrointestinal smooth muscle (Spedding, 1988; Defeudls and Christen, 1989). Since the membrane calcium channel on the parietal cell is of the receptor- operated type (Wolfe and Soli, 1988) which is less sensitive than voltage-operated channels to blockade by calcium channel blockers, and since the tissue selectivity of calcium channel blockers is greater for vascular smooth muscle than for gastrointestinal smooth muscle, it Is unlikely that antiulcer activity can be achieved with the currently available members of this group of calcium antagonists without significantly affecting cardiovascular function (Defeudis and Christen,

1989; Wolfe and Soil, 1988; Spedding, 1988). CHAPTER II

RATIONALE AND AIMS OF THIS STUDY

Although the potent antisecretory agents such as H2 -recep tor

blockers and H*/K+-ATPase inhibitors are highly effective in treating peptic ulcers, there is some concern about the potential development of

gastric carcinoid due to the sustained hypergastrinemia associated with their chronic administration (Creutzfeldt, 1988; Hakanson and Sunder,

198G), and about the frequent relapse ulceration following cessation of their use (Goodwin, 1988; Whittle and Garner, 1988). Moreover, prophylactic use of the H2 -receptor blockers to prevent stress ulcers in patients with severe Illness is associated with a significant increase in

Incidence of nosocomial pneumonia due to bacterial overgrowth in the stomach (Craven et al., 1986; Kleiman et al., 1988). It Is, therefore, necessary to continue developing novel safe and effective antiulcer d r u g s .

Since calcium ions play a pivotal role at various etiological steps in ulcer formation (Wolfe and Soli, 1988), it prompted investigators to examine the potential usefulness of calcium channel blockers in treating peptic ulcers. However, results obtained from both experimental and clinical studies with a variety of calcium channel blockers have been

Inconsistent and conflicting, with antiulcer activity demonstrated only at

79 80

very high doses (Hertz and Cloarec, 1989; Speddlng, 1988; see section

1.6). This is likely due to the uncertainties concerning the existence,

type, or state of voltage-operated calcium channels in the parietal cell

and the gastrointestinal smooth muscle (Defeudis and Christen, 1989;

Spedding, 1988). In order to circumvent the complexities and

uncertainties of the membrane calcium channel, the use of an

intracellular calcium antagonist as a potential antiulcer drug seemed to

provide an attractive alternative. In this respect, the intracellular

calcium antagonist trlmethoxybenzoate (TMB-8) has been shown to

protect rats against reserpine-induced gastric ulceration (Olubadewo,

1988) and inhibit evoked histamine release from rat mast cells In the absence of extracellular calcium (Chakravarti and Yu, 1984). Moreover, magnesium, another intracellular calcium antagonist (Rahwan et al.,

1983), was demonstrated to significantly protect rats against ethanol- and indomethacin-induced gastric lesions (Ghanayem et al., 1987) and inhibit calcium-induced gastric acid secretion (Christiansen et al.,

1975). The present Investigation was undertaken to examine the potential antiulcer activity of propyl-methylenedioxylndene (pr-MDI), an intracellularly-acting calcium antagonist developed In our laboratory

(Rahwan, 1989; Witiak et al., 1974).

In addition to the intraceilualr calcium antagonistic action, it was anticipated the pr-MDI would offer an even better alternative to TMB-8 since pr-MDI also exhibits competitive antagonism of the histamine- activated cardiac H 2 -receptor-coupled adenylate cylcase activity

(Johnson, 1979; Johnson and Grupp, 1979). Moreover, pr-MDI blocks 81 histamlne-lnduced (Hi-receptor mediated) and acetylcholine-induced

(muscarinic receptor-mediated) gastrointestinal smooth muscle contraction (Rahwan et al., 1977). Furthermore, the sensitivity of stimulus-secretion coupling mechanisms in vitro to the inhibitory effects of pr-MDI is 500-1000 times greater than the sensitivity of excitation- contractlon coupling mechanisms (Piascik et al., 1978). If the same holds true In vivo, it should be possible to achieve antiulcer activity with pr-MDI at doses which produce only minimal or no cardiovascular effects, and more than likely also prevent hypergastrinemia.

The aims of the present investigation were four-fold. First, we examined the antiulcer activity of pr-MDI in the cold-restraint stress- induced gastric ulcer model in rats, with the purpose of verifying the assumption that antiulcer activity in vivo would be exhibited at doses lower than those required to produce cardiovascular effects. Second, we examined the antiulcer mechanisms of pr-MDI by investigating its effects on three calcium-dependent ulcerogenic processes: hydrochloric acid secretion, mast cell degranulation, and gastric motility. Third, since duodenal ulcer is 4 times more common than gastric ulcer in the United

States (Richardson, 1989), we also examined the potential antiulcer activity of pr-MDI in the cysteamine-induced duodenal ulcer model in rats. Fourth, since TRH was proposed to be the brain mediator responsible for cold-restraint stress gastric ulcers in rats (Tache et al., 1989), we examined the inhibitory actions of pr-MDI on TRH- induced increases in gastric acid secretion, gastric emptying, and gastric ulceration, and attempted to correlate the antiulcer activity of 82 pr-MDI in the cold-restraint stress model and the TRH-induced gastric lesion model. CHAPTER III

ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE. EFFECTS ON COLD-RESTRAINT STRESS-INDUCED ULCERS, ACID SECRETION, MAST CELL DEGRANULATION AND GASTRIC EMPTYING IN RATS.

3.1 Introduction

H 2 -receptor blockers (e.g., cimetidine) are currently the most important drugs for the treatment of peptic ulcers. Despite their efficacy and safety, there is some concern about the possibility of development of gastric carcinoid (so far seen in rats but not in humans) due to the hypergastrinemia associated with their chronic use

(Hakanson and Sunder, 198G; Whittle and Garner, 1988), and about the frequent relapse ulceration following cessation of their use. The search for newer antiulcer drugs (Whittle and Garner, 1988) led to the development of Inhibitors of the parietal cell H+/K+-ATPase exchange pump (e.g., omeprazole), but these drugs did not circumvent the potential hazard of causing gastric carcinoid (Hakanson and Sunder,

198G; Wolfe and Soil, 1988). The recently developed selective muscarinic

Mi-receptor blockers (e.g., pirenzepine), while exhibiting a narrower spectrum of side-effects as compared to the atropine-like nonselective

M1/M 2 -receptor blockers, are nevertheless much weaker antiulcer agents as compared to the latter drugs (Wolfe and Soli, 1988). Prostaglandin

83 84

Ei derivatives (e.g., misoprostol) have not fulfilled their promise

(Hawkey and Walt, 1986; Whittle and Garner, 1988) and find limited use

in the treatment of peptic ucler, while somatostatin analogs (e.g.,

octreotide; Lamberts, 1988) suffer from lack of selectivity and the

inconvenience of parenteral administration.

The continuing search for safe and effective antiulcer drugs led to

examination of the potential usefulness of the organic calcium channel

blockers (Kleiman et al., 1988; Spedding, 1988), based on the pivotal

requirement for calcium at various etiological steps in ulcer formation

(Wolfe and Soli, 1988) Including the release of histamine, acetylcholine,

gastrin, and hydrochloric acid, the contraction of gastric smooth muscle, and the regulation of mucosal blood vessel tone (Grossman,

1976; Spedding, 1988; Wolfe and Soil, 1988). However, the experimental and clinical results with a variety of calcium channel blockers have been inconsistent and conflicting (Kleiman et al., 1988; Spedding, 1988), with antiuicer activity in many instances demonstrable only at very high doses. This is possibly due to the uncertainty concerning the existence, function, type, or state of voltage-dependent calcium channels in parietal cells (Spedding, 1988), and the likelihood that parietal cell membrane calcium channels are of the receptor-operated type (Wolfe and Soil, 1988) which are generally less sensitive to blockade by calcium channel blockers than are voltage-operated channels

(Bolton, 1979). Furthermore, gastrointestinal smooth muscle contractility is less readily inhibited by organic calcium channel blockers than is contraction of vascular smooth muscle or cardiac muscle (Spedding, 85

1988). It Is, therefore, unlikely that antiulcer activity can be achieved with the currently available members of this class of drugs without significantly affecting cardiovascular function.

In order to circumvent the complexities and uncertainties of the membrane calcium channels, the use of an intracelluarly-acting calcium antagonist as a potential antiulcer drug seemed to present an attractive alternative. Propyl-methylenedioxylndene (pr-MDI; for review see

Raliwan, 1989) is a calcium antagonist with antiarrhythmic, vasodilating, and negative inotropic properties, which acts primarily by Inhibiting calcium mobilization from the endoplasmic reticulum (Wong and Ilahwan,

1988, and references therein). In addition to calcium antagonism, three other pharmacological properties of pr-MDI justified our Interest in exploring its potential antiulcer activity: First, pr-MDI exhibits competitive antagonism of the histamine-activated cardiac H 2 -receptor- coupled adenylate cyclase system with a pA 2 of 6.46 as compared to a pAz of 6.1 for cimetidine (Johnson, 1979; Johnson and Grupp, 1979).

Second, pr-MDI blocks histamine-induced (Hi-receptor mediated) and acetylcholine-induced (muscarinic receptor-mediated) gastrointestinal smooth muscle contraction (Rahwan et al., 1977). Third, the sensitivity of stimulus-secretion coupling mechanisms in-vitro to the inhibitory effects of pr-MDI is 500-1000 times greater than the sensitivity of excitation-contraction coupling mechanisms (Piascik et al., 1978), thus offering the possibility of dissociating the potential antiulcer effects from the known cardiovascular actions of the drug. Such a pharmacological profile, coupled with a remarkable lack of toxicity In 8 6

acute and subchronic rodent toxicity studies (Rahwan et al,, 1979),

provided the rationale for exploring the potential antiulcer activity of

pr-MDI in the present study.

The purposes of this phase of the investigation were to establish

the antiulcer activity of pr-DMI In the cold-restraint stress-induced

gastric ulcer model in rats, with the aim of verifying the assumption

that such activity in-vivo would be exhibited at doses lower than those

required to produce cardiovascular effects, and to examine three

potential mechansims for the antiulcer action of pr-MDI: the effects on

hydrochloric acid secretion, on mast cell degranulation and on gastric

m otility.

3.2 Methods and Materials

Male Sprague-Dawley rats (Harlan Industries, Cumberland,

Indiana) weighing 150-270 g were housed in facilities at constant

temperature (70±2)°F and humidity (50±5)%, and with 12-hr light-dark

cycle. Prior to drug experimentation, the rats were placed in

individual cages with raised wire mesh floors (to prevent coprophagy)

and deprived of food for 24 hours. Free access to water was allowed

until one hour before drug administration.

3.2,1 Effect of pr-MDI on cold-restraint stress-induced ulcers

The fasted rats were then treated intraperitoneally (i.p.) with either saline (0.9%) or DMSO (15%) as vehicle controls, pr-MDI (10, 20, and 30 mg/kg; representing l/18th, l/9th, and l/6th of its LDso), 87

verapamil (11, 16, and 32 mg/kg; representing l/6th, l/4th, and l/2th

of its LDso), or cimetidine (10, 20, and 30 mg/kg; representing l/33rd,

l/16th, and 1/llth of its LDso). The rationale for selection of these

doses is presented below (Section 3.4), Ten minutes after drug or

vehicle administration, the rats were immobilized in individual plastic

restrainers (Fisher Sicentific Co., Cincinnati, Ohio, catalogue item

1-280, medium size, with two test tubes Inserted laterally to further

restrict mobility) (Senay and Levine, 1967) and exposed to 4°C for 3

hours in a ventilated cold room in the dark (Glavin, 1988). Within 10

minutes after the cold-restraint stress the rats were decapitated and

the stomachs quickly excised, washed, and opened along the greater

curvature. The open stomachs were rinsed under tap water, and the

mucosa examined for ulcers with the aid of a stereomicroscope (lOx).

The number of ulcers and the cumulative lesion lengths (measured along

the greater diameter) were recorded.

3.2.2 Effect of pr-MDI on gastric acid secretion

The fasted rats were anesthetized with ether (Mallinckrodt, Paris,

Kentucky), and a longitudinal midline incision of 3 cm was made from

the xyphoid-sternum. Pyloric ligation was performed as described by

Shay et al. (1945) by placing a tight silk suture (Ethicon, Size 3-0,

Somerville, N.J.) around the junction between the pylorus and duodenum. Caution was exercised in order to avoid damage to blood vessels or traction of the stomach wall. The abdomen was then closed with intermittent sutures. Immediately after surgery the rats were 8 8 injected i.p. Into the ileac fossa with one of the following: saline

(control), pr-MDI (30 mg/kg), verapamil (16 mg/kg), or cimetidlne (10 mg/kg). The ether anesthetic was then discontinued, and the abdominal surface of the rats was cleaned with saline, dried, and covered with collodion (Mallinckrodt, Paris, Kentucky), Ten minutes after drug adminstratlon, the rats were treated subcutaneously (s.c.) with either saline or bethanechol[3.2 mg/kg (Ogle et al., 1985)], the latter to stimulate hydrochloric acid secretion. The rats were then returned to their individual cages with no food or water for 2 hours. The animals were subsequently killed with ether overdose, and the stomachs removed after an additional ligature was placed around the esophago- cardiac junction. The Isolated stomachs were rinsed in saline and blotted dry, and a puncture was made in the rumenal segment along the greater curvature. The gastric content of individual stomach was collected in graduated centrifuge tubes and centrifuged at 1,500 xg for

20 minutes (IEC HN-SII centrifuge, Damon/IEC Division, Needham Hts.,

Massachusett). The volume of clear gastric content was measured and expressed as ml/100 g body weight/hr. The pH was recorded (Corning pH-meter, Model 7, Madfield, Massachusett), and the total acid output was assessed by titration against 0.1 N NaOH to pH 7.0 and expressed as uEq/100 g body weight/hr.

3.2.3 Effect of pr-MDI on mast cell degranulation

This part of the experiment was conducted simultaneously with the gastric acid secretion study (see section 3.2.2), As soon as the 89

procedure of gastric contents collection was completed, the stomach was

opened along Its greater curvature, A small piece of tissue block (1cm

x 1cm) was excised from the glandular portion of the stomach and fixed

In 10% formalin solution for at least 24 hours. The samples were sent to

the pathobiology laboratory at the College of Veterinary Medicine for

histological processing. The tissue samples were dehydrated with

ethanol (80-100)%, cleared with xylene, and embedded in paraffin.

Sections of 6 u thick were made by cutting the paraffin blocks

vertically to the mucosal surface, and stanined metachromatlcally with

0.5% (w /v ) aqueous toluidine b lu e.

The mast cell counts were performed with a binocular Zeiss

microscope In an area immediately below and parallel to the mucosal

surface epithelium (mucosal count) and in the submucosa (submucosal

count) for 40 consecutive oil immersion fields (o.i.f.) (lOOOx) (Cho and

Ogle, 1978; Guth and Hall, 1966). The total area counted at each level

was 0.8 mm2 . The results were expressed as number of granulated

metachromatically stained mast cells per 40 o.i.f.

3.2.4 Effect of pr-MDI on gastric emptying

The method of Scarpignato et al. (1980) and of Brage et al.

(1986), with minor modifications, was used to examine the effect of pr-

MDI on gastric emptying. This method Is considered the most direct

approach for assessment of drug-induced changes in motility (Walsh,

1989), although an effect of the drug on the pyloric sphincter cannot be excluded. Fasted rats (as described above) were pretreated i.p. with pr-MDI (10, 20, or 30 mg/kg), verapamil (16 mg/kg), or saline

(basal gastric emptying), 30 minutes before the oral administration of

1.5 ml of a methylcellulose/phenol red meal (see below) by means of an oral gavage needle. One hour after the meal, the test animals were

sacrificed by ether overdose. A separate group of saline-pretreated rats

(controls) was sacrificed immediately after administration of the test meal, and the data obtained from this group (representing 0% gastric emptying) was used for the calculations shown below. After sacrifice and laparotomy, both the pylorus and the cardia of each stomach were ligated, and each stomach was removed and placed in a beaker containing 5 ml of 0.1 N NaOH. Each stomach was then minced and homogenized (TIssumizer®, Tekmar, Cincinnati, Ohio). The homogenates were centrifuged at 5,000 xg for 5 minutes at 4°C (Sorvall superspeed centrifuge, Model RC2-B, Newton, Connecticut). To each supernatant was added 0.5 ml of 20% trichloroacetic acid to precipitate the proteins, followed by centrifugation at 1,500 xg for 15 minutes using the IEC HN-

SII centrifuge. The supernatant obtained from this centrifugation was then mixed with 1 ml of 1 N NaOH to maximize the color intensity. An aliquot (1 ml) of the sample was diluted in 2 ml of distilled water, and the absorbance of the colored sample was measured at 560 nm with a

Spectronic 20 spectrophotometer (Bausch and Lomb Inc., Rochester,

N .Y .). The absorbance of samples derived from test animals sacrificed one hour after the meal was calculated as a percentage of that of the controls (sacrificed immediately after the meal and considered as 100% absorbance or 0% gastric emptying). The percent gastric emptying 91

(G.E.) was therefore calculated as follows:

absorbance of sample of test animal G .E , (%) ~ (1 ...... - ...... ) x 100% absorbance of sample of control animal

The methylcellulose/plienol red meal was prepared by adding 50 mg of

phenol red (as a nonabsorbable marker) to 100 ml of 1.5% (w/v)

methylcellulose solution at 80°C with constant stirring. The meal was

allowed to cool down to 35°C before oral intubation.

3.2.5 Materials and data analysis

Verapamil HC1, cimetidine, bethanechol, and methylcellulose were

purchased from Sigma Chemical Co. (St. Louis, Missouri), and phenol red from Coleman and Bell Co. (Norwood, Ohio). Pr-MDI HC1 was

custom synthesized at Chem Biochem Research Inc. (Salt Lake City,

Utah). All drug solutions were freshly prepared before use, and administered in a volume of 2 ml/kg body weight. Verapamil, Pr-MDI, and the lowest dose of cimetidine were dissolved In saline, while the intermediate and highest doses of cimetidine were dissolved in DMSO and diluted with saline (15% DMSO final concentration). The data are

presented as the mean±S.E.M. The unpaired Student t test was used

for statistical analysis (Sinclair, 1988), and significance was set at p=0.05 or better. A regression analysis was performed to determined the correlation coefficient (r) between % inhibition of ulcer formation and % delay in gastric emptying produced by pr-MDI. 92

3.3 Results

3.3.1 Effect of pr-MDI on cold-restraint stress-induced ulcers

The ulcers arising from the cold-restraint stress were exclusively localized in the glandular portion of the stomachs of the rats. They were manifested as black focal hemorrhagic erosions and elongated hemorrhagic streaks along the ridges of the mucosal folds , with sharply demarcated edges (Figure 20).

Table 1 represents the prophylactic antiulcer effects of pr-MDI, verapamil, and cimetidine. The vehicle control groups (saline and

DMSO) did not differ from each other, with the mean number of ulcers for these groups being 14.610.9G and 15,3711.79. The corresponding mean cumulative length of ulcerated stomach surface was 16.3811.83 mm for the saline group and 12.1611.57 mm for the DMSO controls. Thus,

DMSO used to solubilize the higher doses of cimetidine had no influence on stress-induced ulcers. Gross examination of the stomachs of the vehicle control animals revealed the stomach wall to be rigorously contracted with massive bleeding and submucosal hyperemia. None of the control animals died during the cold-restraint stress.

Pr-MDI, at doses of 10, 20, and 30 mg/kg, afforded dose- dependent protection against the stress-induced gastric ulcer, with the mean number of ulcers declining to 9.8711.97, 5.2810.92, and 2.3310.53, in the ascending order of the doses administered. The corresponding mean cumulative length of ulcerated stomach surface also decreased dose Figure 20. The effect of pr-MDI on stress-induced ulcers in rots. Representative examples of stomachs from saline-treated controls (A) and (B), and rats treated with 10 mg/kg (C) and 30 mg/kg (D) pr-MDI. 9 4

Table 1. Effects of pr—HDI, verapamil, and cimetidine on cold-restraint stress ulcer formation in rats

Treatment Dose(mg/kg) No. o f rats No. o f u lcers Ulcer Length(mm)

S alin e — 15 14.60±0.96 16.38±1.B3

DMSO — 6 15.37±1.79 12.16±1.57

Pr—HDI 10 8 9.87±1.97* 8.75±1.82*

20 7 5.28±0.92*** 6.24+1.82**

30 9 2.33+0.53*** 1.62±0.47***

Verapamil 11 5 8.40±3.12* 6.96±2.69*

16 6 5.00+1.15*** 4.18±1.10***

32 6 0.83+0.48*** 0.68±0.46***

Cimetidine 10 6 3.00±0.84*** 2.41±0.83***

20 7 4 .00±0.95*** 1.96+0.65***

30 7 1.00±0.44*** 0 .78±0.41***

QValues represent the meanirSEH *P<0.05; **P<0.01; ***P<0.001; when compared to sa lin e — or DHSCHtreated r a t s . 95

dependenlly to 8.75+1.82 mm, 6.2411.82 mm, and 1.6210.47 mm. Gross

examination of the stomachs revealed that the stomach wall was relaxed, and distended due to retention of brownish-colored gastric fluid contents. None of the pr-MDI-treated rats at any dose died during the cold-restraint stress.

Verapamil also significantly protected the rats against cold- restraint stress-induced ulcers in a dose-dependent manner. Verapamil doses of 11, 16, and 32 mg/kg reduced the ulcer incidence to 8.413.12,

5.011.15, and 0.8310.48, respectively, and the corresponding mean cumulative length of ulcerated stomach surface was reduced to 6.9612.69 mm, 4.1811.10 mm, and 0.6810.46 mm. Gross appearance of the stomachs in verapamil-treated rats was similar to that of pr-MDI-treated rats.

However, the mortality rate due to verapamil pretreatment was quite high, especially at the higher doses, and approximated 23% of the anim als,

Although cimetidine significantly inhibited ulcerogenesis, the effect was not dose-dependent. Doses of 10, 20, and 30 mg/kg reduced the ulcer incidence to 3.010.84, 4.010.95, and 1.010.44, respectively. The corresponding mean cumulative length of ulcerated surface was reduced to 2.4110,83 mm, 1.9610.65 mm, and 0.7810.41 mm. On gross examination the stomach wall was contracted and there was no remarkable hemorrhagic contents. None of the cimetidine-treated rats died during the cold-restraint stress. 9 6

3.3.2 Effect of pr-MDI on gastric acid secretion

The effects of pr-MDI, verapamil, and cimetdine on bethanechol - stimulated gastric acid secretion in pylorus-ligated conscious rats are shown in Table 2. The 2-hour ligation did not result in ulcers. As compared to unstimulated rats, bethanechol significantly increased gastric volume (PO.OOl), titratable acidity (P<0.05) and total acid output (P<0.001), and reduced the pH of the gastric contents (P<0.01).

Cimetidine (10 mg/kg; used as a positive control) significantly obtunded the reduction in pH (P<0.001), and the increases in titratable acidity

(P<0.001) and total acid output (P<0,001) of the gastric contents induced by bethanechol, but did not influence the volume of gastric contents. Verapamil at 16 mg/kg, a dose which produces the same antiulcer activity as 20 mg/kg pr-MDI, likewise significantly obtunded the reduction in pH (P<0.001), and the increases In titratable acidity

(P<0,001) and total acid output (P<0.001) of the gastric contents induced by bethanechol, but also obtunded the increase in the volume of gastric contents (P<0,01), Verapamil and cimetidine (at doses indicated above) were equleffective in antagonizing the increase in total acid output of gastric contents induced by bethanechol. Pr-MDI given at 30 mg/kg, a dose which exhibits greater antiulcer activity than 16

verapamil, while significantly obtunding the reduction in pH

(P<0.01) and the increase in total acid output (P<0.05) of the gastric contents induced by bethanechol, was significantly less effective in this respect as compared to both verapamil and cimetidine (P<0.01 or better) . Pr-MDI did not significantly obtund the increases in titratable Table 2. Effects of pr—HDI, Verapamil and cimetidine on bethanechol-induced gastric acid secretion.

No. of G astric Vol Total Acidity Treatment Secretagogue Rat (ml/lOQg/hr) pll (uEq/lOOg/hr)

Saline Saline 6 1.21±0.16*** 1.71+0.13** 73.02112.26***

Saline Bethanechol 11 2.13+0'. 12 1.35+0.02 189.25112.93 (3.2 mg/kg)

Pr-MDI Bethanechol 8 1.83+0.13 1.50+0.04** 141.71110.18* (30 mg/kg) (3.2 mg/kg)

Verapamil Bethanechol 8 1.5210.17** 1,7910.05*** 71.5317.40*** (16 mg/kg) (3.2 mg/kg)

Cimetidine Bethanchol 5 2.29±0.09 2.2110.23*** 84.37110.49*** (10 mg/kg) (3.2 mg/kg)

*P<0.05. **P<0.01, *P<0.001 as compared to saline/bethanechol-treated group. 98 acidity (only a modest 12% inhibition) and volume of gastric contents induced by bethanechol.

3.3.3 Effect of pr-MDI on mast cell degranulation

The morphology of the granulated metachromatically stained mucosal mast cells and submucosal (connective tissue) mast cells is depicted in

Figure 21. Metachormasia refers to the stained granules of mast cells taking on different color (purple-red) from that of the applied dye

(toluidine blue). Mucosal mast cells are generally smaller, and contain fewer granules of more variable shape, while submucosal mast cells are larger in size with more granules of even shape. As a result, the mucosal mast cells were stained lighter than the submucosal mast cells.

The effects of pr-MDI and verapamil on mast cell counts at the mucosal and submucosal areas of the stomach wall in rats stimulated with bethanechol are summarized in Table 3. However, the data demonstrates low mucosal and submucosal mast cell counts and there is no difference identified between the saline-treated (control) group and the bethanechol-treated (mast cell depletion) group. In the present study, the mucosal mast cell count in the saline-treated group was 5-10 times lower than that of other published results, while submucosal mast cell count was at least 2 times less than other reports (Cho and Ogle,

1978; Guth and Hall, 1966; Hasanen, 1960), Failure of this experiment indicates some procedural errors occurring during histological processing which will be explained In discussed below (Section 3.4). 9 9

Figure 21, Photomicrographs of a section of the gastric mucosa (top) and the gastric submucosa (bottom). Observe the metachromatically stained (purple color) mucosal mast cells at the surface of the mucosa (top) and submucosal mast cells in the loose connective tissue of the submucosa (bottom). 100

Table 3. Effect of pr—MDI and verapamil on mast cell counts at mucosal and submucosal area of the stomach of rats treated with 3.2 mg/kg bethanechol subcutaneously.

Mast C ell 1Counts (40 o . i . f . No. of Treatment Secretagogue Rats Mucosal Submucosal

Saline Saline 5 26.60±4.98 27.8011.91

Saline Bethanechol 7 31.71±6.16 33.7114.93

Pr-MDI Bethanechol 7 32.8614.40 32.5713.62 (30 mg/kg)

Verapamil Bethanechol 5 36.4019.87 29.2013.10 (16 mg/kg)

*40 o .i.f. equals to 0.8mmz total area of counting. 101

3.3.4 Effect of pr-MDI on gastric emptying

The effects of pr-MDI and verapamil on gastric motility, measured as gastric emptying, are shown in Table 4. Gastric emptying recorded one hour after the methylcellulose meal in saline-pretreated rats was almost complete (83%). While verapamil (1G mg/kg) and pr-MDI (20 mg/kg) produce equivalent antiulcer effects, significant delay of meal- induced gastric emptying was produced only by pr-MDI (P<0.01). An even greater slowing of gastric emptying resulted from pretreatment with 30 mg/kg pr-MDI (P<0.001). On the other hand, the delay of gastric emptying induced by 10 mg/kg pr-MDI was not significant, although this dose significantly inhibits stress-induced gastric ulceration.

When % inhibition of stress-induced ulceration In rats (Table 1) was plotted against the % delay in gastric emptying (Table 4) induced by pr-MDI, a close relationship between the two variables was evident from a correlation coefficient (r) of 0.997 (P=0.05) (Figure 22).

3.4 Discussion

Although It Is often stated that the evidence for hyperacidity as a cause of human duodenal ulcer is marginal and for gastric ulcer Is nonexistent (Bass et al., 1989), yet It has rationally been argued that failure to demonstrate luminal hyperacidity may be due to loss of H+ by back-diffusion through the damaged gastric mucosal barrier (Bowman and Rand, 1980). Furthermore, normal or even subnormal levels of 102

Table 4. Effect of pr-MDI and verapamil on gastric emptying of rats treated with test meal for 1—hr.

Treatment N G astric Emptying (%)

Saline(control)* 5 100 Saline(basal) 5 8 3 .24±3.04

Verapamil(16mg/kg) 5 79,52+7.37

Pr-MDI(lOmg/kg) 5 81.98±6.99 Pr-MDI(20mg/kg) 4 6 3 .18±5.15** Pr—MDI( 30mg/kg) 5 37.76±7.22***

*Control rats were killed immediately after oral feeding of the test meal. **p<0.01; ***p<0.001 as compared to the basal values. g e 2 A eainhp ewen Ihbto o sres-nduced gastric d e c u d ss-in e str of % Inhibition een betw relationship A 22. re igu F % % DELAY IN GASTRIC EMPTYING 0 2 - 0 3 “ 0 4 50 - 50 -1 0 6 - le ad ihbto o gsrc mpyn i rt by rMDI. pr-M y b rats in ptying em gastric of % inhibition and ulcer aa s ae fo Tals ad 4. and 2 ables T from taken as w Data 0.05 0.997 % % INHIBITION OF STRESS-INDUCED ULCER 100 103

104

stomach acid and pepsin may be sufficient to cause ulceration of a

gastric mucosa which had been damaged by bile reflux from the

duodenum (Greenberger, 198G). Hydrochloric acid secretion results from

combined cholinergic, gastrinergic, and histamlnergic influences on the

parietal cells of the stomach (Black and Shankley, 1987; Wolfe and Soil,

1988). These Influences are exerted, respectively, on cholinergic Mi

(neuronal) and M2 (parietal) receptors, gastrin receptors (parietal),

and histamlnergic H2 receptors (parietal), resulting ultimately in

stimulation of the parietal cell H*/K+-ATPase exchange pump involved in

the terminal step of hydrochloric acid secretion. The release of

histamine (Beaven et al., 1987), acetylcholine (Wolfe and Soil, 1988),

gastrin (Grossman, 1976), and hydrochloric acid (Spedding, 1988) are

all calcium-dependent. Controversy exists about whether acid secretion

from the parietal cell is dependent upon the influx of extracellular

calcium or the mobilization of intracellular calcium (Chew and Brown,

1986), and any influx of extracellular calcium appears to occur through

receptor-operated calcium channels (Wolfe and Soli, 1988), Histamine

release is dependent upon both extracellular and intracellular calcium

(Chakravarti and Yu, 1984; Chakravarti, 1988), and in the rat fundic

mucosa histamine is stored in mucosal mast cells and endocrine-like cells

that possess acetylcholine and gastrin receptors (Nylander et al., 1985;

Enerback, 1985). The extent to which the stimulation of acid secretion

by acetylcholine and gastrin reflects the stimulation of histamine release, as opposed to direct interaction with parietal cell receptors, remains controversial (Wolfe and Soil, 1988). 105

The cold-restraint stress model for ulcer production has been

widely used for studying antiulcer activity of drugs (Pare and Glavin,

1986). Hydrochloric acid secretion is increased, unchanged, or decreased in this model system, but high amplitude contractions of long duration occur in the stomach wall and are considered a major contributing factor to cold-restraint stress-induced ulcers in rats

(Garrick et al., 1986, and references therein). Gastric muscular contraction mediated through activation of Hi receptors by histamine released during cold-restraint stress (Cho and Ogle, 1978) may result in mechanical restriction of mucosal blood flow, leading to mucosal ischemia and ulceration (Garrick et al., 1986). Furthermore, the released histamine may cause mucosal microcirculatory congestion by an action on H2 receptors, thereby contributing to ischemia and ulceration

(Lau and Ogle, 1981).

Cimetidine was used in the present study as a positive control. As anticipated, doses of 10, 20, and 30 mg/kg significantly protected rats against cold-restraint stress ulcers. Lau and Ogle (1981) demonstrated th at i.p. cimetidine doses of 10 mg/kg or greater decreased acid secretion and ulceration, while a dose of 5 mg/kg exhibited antiulcer activity by inhibiting H2 -mediated mucosal microcirculatory congestion without reducing acid secretion.

Pr-MDI produced a dose-dependent prophylactic effect against the development of cold-restraint stress-induced ulcers. The doses (10-30 m g /k g I.p.) which protected the rats against ulceration are 106 considerably lower than those required to demonstrate antiarrhythmlc activity in rodents. The latter effect is observed at ED so values of

53-84 mg/kg i.p. (Lynch et al., 1982). This is in agreement with in- vitro findings indicating a greater sensitivity of stimulus-secretion coupling mechanisms than of excitatlon-contraction coupling mechanisms to the inhibitory actions of pr-MDI (Piasclk et al., 1978). Thus, it is possible to achieve antiulcer activity with pr-MDI doses considerably lower than those required to affect the cardiovascular system.

Verapamil also protected rats against stress ulcers at the high doses used in the present study (11-32 mg/kg i.p.), which most certainly would have pronounced cardiovascular and nonspecific effects.

The doses of 16 and 32 mg/kg used in the present study are ones also used by Glavin (1988). The lowest dose of verapamil (11 mg/kg) corresponds to the highest dose of pr-MDI (30 mg/kg), in that both of these doses represent l/6th of their respective i.p. LDso values in rats

- these being 175 mg/kg for pr-MDI (Rahwan et al., 1979) and 67 mg/kg for verapamil (Hass and Hartfelder, 1962). The equivalent antiulcer activity of pr-MDI and verapamil is achieved at l/9th of the

LDso dose of pr-MDI (20 mg/kg) and l/4th of the LDso dose of verapamil (16 mg/kg). At l/16th (10 mg/kg) of itsLDso, pr-MDI afforded significant protection against ulceration. It Is unlikely that th e H 2 -blocking property of pr-MDI [pAa =6.4G in the Ha-coupled cardiac adenylate cyclase assay (Johnson and Grupp, 1979)] is the sole or major determinant of the difference between the antiulcer effect of this intracellular calcium antagonist and that of verapamil, since the latter 107 calcium channel blocker also exhibits H: receptor blocking activity under the same conditions (pA2=5.44) (Johnson and Grupp, 1979). It is, of course, possible that the cardiac and gastric H2 receptor may respond differently to these drugs.

Although 30 mg/kg pr-MDI significantly obtunded the bethanechol- induced reduction in pH and elevation in total acidity of the gastric contents (Table 2), these effects were significantly weaker (P<0.01 or better) than those produced by both verapamil and cimetidine. It is, therefore, unlikely that the reduction in acid secretion observed with pr-MDI plays a major role in its antiulcer action since at the doses used pr-MDI exerts better antiulcer activity than verapamil (Table 1). It is believed that the intracellular calcium antagonistic action of pr-MDI cannot be fully expressed in the parietal cell since this type of cell has only scant smooth endoplasmic reticulum, which is the Identified target for the intracellular inhibitory action of pr-MDI on calcium mobilization

(Wong and Rahwan, 1988). While the reduction in acid secretion produced by cimetidine (10 mg/kg) may reflect its H2 -b lock in g property, it Is likely that the similar reduction in acid secretion produced by the very high dose of verapamil used (16 mg/kg) may reflect an inhibition (probably nonspecific) of the H+/K+-ATPase pump rather than a selective action on calcium channels (Herling and

Ljungstrom, 1988).

In a previous study (Rahwan et al., 1977) pr-MDI was shown to block acetylcholine-induced contractions of the rat ileum and histamine- 108 induced contractions of the guinea pig ileum in-vitro. The present investigation extends these findings in-vivo by demonstrating a dose- dependent inhibition of gastric emptying [used as an index for gastric motility (Walsh, 1989)] . At 20 and 30 mg/kg doses of pr-MDI the inhibition of gastric emptying was significant (P<0.01 or better) (Table

4). Verapamil, on the other hand, even at the high dose of 16 mg/kg used, had no effect on gastric emptying. The inhibitory effect of pr-

MDI on gastric emptying (Table 4) correlated well with its antiulcer activity (Table 1), as evidenced by a correlation coefficient close to unity (Figure 20). These findings are taken as evidence that inhibition of gastric smooth muscle contraction, mediated by the intracellular calcium antagonistic action of pr-MDI, may well represent a major mechanism for the antiulcer action of this drug. This is in line with the findings of Garrick et al. (1986) who demonstrated that the smooth muscle relaxant, papaverine, Inhibits the high amplitude gastric contractions and the ulcers associated with cold-restraint stress without altering acid secretion. That other mechanisms may be involved in the antiulcer action of pr-MDI (e.g. Hz-antagonism, antlsecretory effect, or other as yet unidentified mechanisms) is evident from the fact that the

10 mg/kg dose exhibits significant antiulcer activity (Table 1) but reduces gastric motility minimally (Table 4).

The modest (but significant) reduction by pr-MDI of the gastric acid secretion evoked by the cholinergic muscarinic agonist, bethanechol

(Table 2), may be a consequence of the reduction of gastric motility by pr-MDI (Table 4). Indeed, increased motor activity of the stomach is 109 frequently associated with increased acid and pepsin secretion, mediated by a heightened vago-vagai reflex pathway (Greenwood and Davison,

1987).

It is not clear why 16 mg/kg verapamil failed to attenuate gastric emptying (Table 4) when its calcium channel blocking activity would have predicted otherwise. Nevertheless, Brage et al. (198G), u sin g the same experimental approach described here, reported that the EDso of verapamil for Inhibiting gastric emptying in the rat is 21.4 mg/kgi.p., which is higher than the dose of verapamil used in the present study.

Such high doses of a calcium channel blocker would certainly exert profound cardiovascular and toxic effects and preclude any consideration of potential usefulness as an antiulcer agents.

It has been suggested that cold-restraint stress ulcer, which is primarily mediated by enhanced cholinergic activation, is associated with increased stomach mast cell degranulation causing the release of histamine and thus producing gastric hypermotility and microcirculatory vasocongestion (Cho and Ogle, 1978). Verapamil has been shown to reverse the decrease in mucosal and submucosal mast cell counts induced by bethanechol (Ogle et al., 1905), and TMB-8 and magnesium [two intracellular calcium antagonists (Chiou and Malagodi,

1975; Rahwan et al., 1973)] also exhibit antiulcer activity in rats

(Olubadewo, 1980; Ghanayem et al., 1987)' and Inhibit evoked histamine release from rat mast cells in the absence of extracellular calcium

(Chakravarti and Yu, 1984). It is therefore possible that the antiulcer 110

action of pr-MDI may be due to Inhibition of intracellular calcium-

dependent release of histamine, leading to gastric smooth muscle relaxation and promotion of gastric mucosal blood flow. However, the

present investigation unfortunately failed to verify this aspect of

antiulcer action of pr-MDI due to errors occurring during histological

processing of the gastric samples. Instead of fixing the gastric tissue

in 4% lead acetate solution or absolute methanol solution, the samples obtained from the present experiment were fixed in regular 10% formalin aqueous solution which has been shown to degranulate mucosal cells resulting in dramatic decrease in both mucosal and submucosal mast cell counts (Enerback, 1985; Guth and Hall, 1966; Rasanen, 19G0).

Moreover, formalin fixation was found to block the cationic dyes (e.g., toluidine blue) binding to mucosal mast cell granules which contain

sulphated glycosaminoglycan (Enerback, 1985), and produce

substantially lower metachromatic staining for mucosal mast cells.

In conclusion, the results of the present investigation demonstrate that pr-MDI can exert significant antiulcer activity at doses (10-30 mg/kg) substantially lower than those producing antiarrhythmic effects[EDso = 68 mg/kg I.p. (Lynch et al., 1982)] . In view of the remarkable lack of toxicity of pr-MDI in acute and subchronic toxicity studies in rodents (Rahwan et al., 1979), further development of this drug as a potentially effective and safe antiulcer agent is deemed warranted. Pr-MDI would offer an advantage over Ha blockers and inhibitors of the H*/K*-ATPase pump If it can be demonstrated that the antisecretory properties of this calcium antagonist produce I ll hypogastrinemia rather than hypergastrlnemla upon chronic administration, and if its antiulcer activity can be demonstrated upon oral administration. Being a tertiary amine hydrochloride, pr-MDI should be readily absorbed from the small intestine (D.T. Witiak, personal communication). CHAPTER IV

ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE. EFFECT ON CYSTEAMINE-INDUCED DUODENAL ULCERS IN RATS.

4.1 Introduction

The mechanisms involved In peptic ulceration are multifactorlal,

and represent a breakdown In the balance between acid-pepsin secretion

and mucosal defense mechanism (Eass et al., 1989). Current approaches

to the treatment of peptic ulcers Involve H 2 -receptor blockers,

HVK*-ATPbsb inhibitors, antacids, sucralfate, and muscarinic-receptor

blockers. Calcium channel blockers have been tried In the treatment of

peptic ulcers because of the pivotal requirement for calcium ions at

various etiological steps in the development of these ulcers (Wolfe and

Soli, 1988), including the release of histamine, acetylcholine, gastrin, and hydrochloric acid, the contraction of gastrointestinal smooth muscle, and the regulation of mucosal blood vessel tone (Grossman, 1976;

Spedding, 1988; Wolfe and Soil, 1988), However, the experimental and clinical results with a variety of calcium channel blockers have been inconsistent and conflicting (Kleiman et al., 1988; Spedding, 1988), and antiulcer activity in many instances was demonstrable only at very high doses which would adversely affect the cardiovascular system. This is possibly due to the uncertainty concerning the existence or type of

112 113 membrane calcium channels in parietal cells (Spedding, 1988; Wolfe and

Soil, 1988), and the lower sensitivity of calcium channels in gastrointestinal smooth muscle to calcium channel blockers as compared to calcium channels in vascular smooth muscle and cardiac muscle

(Spedding, 1988).

In an attempt to circumvent the complexities and uncertainties of the gastrointestinal membrane calcium channels, we examined the potential nntiulcer activity of propyl-methylenedioxyindene (pr-MDI), an intracellular-acting calcium antagonist (Rahwan, 1989) which inhibits calcium mobilization from the endoplasmic reticulum (Wong and Rahwan,

1988). This calcium antagonist also blocks Ha-receptors (Johnson,

1979; Johnson and Grupp, 1979) and inhibits Hi-receptor-mediated and muscarinic-receptor-mediated intestinal smooth muscle contraction

(Rahwan et al., 1977). Our findings demonstrated the ability of pr-MDI to prevent the development of cold-restraint stress-induced gastric ulcers in rats in a dose-dependent manner at doses (10-30 mg/kg; i.p.) significantly lower than those required to exhibit antiarrhythmic activity

(see Chapter III), The inhibitory effect of pr-MDI on the development of gastric stress ulcers was highly correlated with its inhibitory effect on gastric motor activity, with only a modest contribution from reduction of gastric acid concentration (see Chapter III) .

Due to the encouraging results witli pr-MDI in the prevention of gastric stress ulcers, the present phase of this investigation was undertaken to explore the potential extension of these results to 114 duodenal ulcers. Duodenal ulcers produced by cysteamine in rats was used as the experimental system, since this model is a simple and reliable method for testing potential antiulcer agents (Selye and Szabo,

1973; Szabo, 1978). The pathogenesis of cysteamine-induced duodenal ulcers include increased (Ishii et al., 1976; Szabo et al., 1977; 1979) or unchanged (Robert et al., 1974) gastric acid concentration, increased duodenal motility (Mangla et al., 1989; Takeuchl et al., 1987), delayed gastric emptying (Lichtenberger etal., 1977; Tanaka et al., 1989), and decreased duodenal bicarbonate secretion In response to acid (Briden et al., 1985; Ohe et al., 1988).

4.2 Methods and Materials

Female Sprague-Dawley rats (Harlan Industries, Cumberland,

Indiana) weighing 180-260 g were housed in facilities at constant temperature (70±2)°F and humidity (50+5)%, and with 12-hour light-dark cycle. Unless otherwise stated, the animals were maintained on Purina

Lab Chow and tap water ad. libitum throughout the experiment.

4.2.1 Cysteamine-induced duodenal ulcers

The method of Robert et al. (1974) was used with minor modifications. Groups of rats received one of the following treatments three times dally at 8:00, 14:00, and 20:00 hr: saline (I.p., co n tro l), atropine (10 mg/kg, s.c.), dopamine (30 mg/kg, i.p.), pr-MDI (10 and

30 m g /k g , i.p.), verapamil (7 mg/kg, i.p.), or the antacid Maalox (1 and 2 ml, p.o.). In one study, Maalox (2 ml, p.o.) was administered at 8:00, 10:00, 12:00, 14:00 and 20:00 hr. The rationale for selection of drugs and doses Is given below. Immediately after the Initial drug treatments, cysteamine hydrochloride (425 mg/kg, s.c.) was administered once at 8:00 hr. Injection volumes were 2 ml/kg for all treatments. Animals were sacrificed in carbon dioxide atmosphere 24 hours after cysteamine adminstration. The stomach and duodenum were excised as a single unit, and opened along the greater cruvature side of the stomach and the mesenteric side of the duodenum. The gastric contents were completely removed, and the opened organ was rinsed in cold saline solution. The mucosa was then examine for incidence and severity of ulceration under a stereomicroscope (10x). Severity was evaluated on a scale of 0-3, where 0 represents normal mucosa, 1 represents superficial erosion, 2 represents deep ulceration with transmucosal necrosis, and 3 represents perforation. The duodenum from each animal was then assigned the severity score corresponding to the severest lesion identified in that duodenum (Robert et al., 1974).

Drug selection and dosage were based on the following published observations: atropine (10 mg/kg, s.c.) reduces the incidence of cysteamine-induced duodenal ulcers in rats by abolishing gastric acid secretion (Takeuchl et al., 1987). Dopamine (30 mg/kg, i.p.) red u ces the severity of cysteamine-induced duodenal ulcers in rats by abolishing duodenal hypermotility without significantly affecting acid secretion

(Takeuchl et al., 1987). Pr-MDI (10 and 30 mg/kg, i.p.) in h ib its stress-induced gastric ulcers in rats by inhibiting gastric motor activity with only a modest inhibition of acid secretion (see Chapter III). 116

Verapamil (7 mg/kg, i.p.)* a calcium channel blocker, was used with the intent of inhibiting gastric acid secretion (see Results section) without influencing gastric motility. Higher doses of verapamil (11-32 m g /k g , i.p.) were previously shown to inhibit gastric stress ulcers in rats, and the dose of 16 mg/kg was shown to inhibit gastric acid secretion without influencing gastric motility (see Chapter III). T he commercial antacid Maalox (which contains 40 mg magnesium hydroxide and 45 mg aluminum hydroxide per ml) was used since a similar commercial antacid was shown to prevent duodenal ulcer formation induced by cysteamine in rats (Robert et al., 1974). Our initial experiments with Maalox involved administration of 1 or 2 ml at 8:00,

14:00, and 20:00 hr, representing 0, 6, and 12 hours following cysteamine administration. Since the neutralizing action of antacids in the non-fasted state lasts up to 3 hours (Fordtran, 1973), and since the reported peak gastric acid secretion evoked by cysteamine is attained 4 hours after the cysteamine administration (Ishii et al., 1976;

Szabo et al., 1977), Maalox (2 ml) was additionally administered in another group of rats at 8:00, 10:00, 12:00, 14:00, and 20:00 hr, representing 0, 2, 4, 6, and 12 hours following cysteamine administration. The ulcerogenic dose of cysteamine employed (425 m g /k g , s.c.) was that of Robert et al. (1974) who reported a s.c.

EDso of 325 mg/kg for the induction of duodenal ulcers in female non- fasted Sprague-Dawley rats. Although the reported s.c. LDso for cysteamine is 450 mg/kg in male rats (Okabe etal., 1982), Szabo (1978) reported a mortality of 20% in female non-fasted rats given 400 mg/kg 117 s.c. In the present study, using female non-fasted rats, 425 mg/kg cysteamine s.c. did not result in deaths. All drug doses mentioned are for the salts (cysteamine HC1, pr-MDI HC1, atropine sulfate, dopamine

HC1, and verapamil HCI).

4.2.2 Gastric acid secretion

Female Sprague-Dawley rats were fasted for 24 hours in cages with raised wire mesh floors (to prevent coprophagy), and allowed free access to water until 1 hour prior to experimentation. Pyloric ligation was performed as previously described (see Chapter III). Briefly, the rats were anesthetized with ether, and a longitudinal midline incision of

2 cm was made from the xyphoid-sternum. The pylorus was ligated by placing a tight silk suture around the junction between the pylorus and the duodenum. The abdomen was then closed with intermittent sutures, and the ether anesthetic was discontinued. Immediately after surgery, the rats were treated with either saline (i.p.), atropine (10 mg/kg, s.c,), verapamil (7 mg/kg, i.p.), or Maalox (1 and 2 ml, p.o.). Ten minutes after drug administration, the rats were injected s.c. with either saline (control), cysteamine[425 mg/kg, (Robert etal., 1974)], or bethanechol[3.2 mg/kg, (Ogle etal., 1985)] in order to stimulate gastric acid secretion. The rats were then returned to their cages with no food or water for 4 hours for the cysteamine groups (representing the time of peak cysteamine-induced acid secretion reported by Szabo et al. (1977) and by Takeuchl et al. (1987),and for 2 hours for the bethanechol-treated groups (Ogle et al., 1985). The animals were 118 subsequently killed with ether overdose, and the stomach removed after an additional ligature was placed around the esophago-cardiac junction.

A puncture was made in the rumenal segment along the greater curvature, and gastric contents were collected and centrifuged. The volume of gastric secretion (supernatant) was measured and expressed as ml/100 g body weight/hr. The pH was recorded, and the acid concentration assessed by titration against 0.1 N NaOH to pH 7.0 using an autoburette (Radiometer, Copenhagen) and expressed as mEq/ml.

Total acid output was calculated as the product of the volume of gastric secretion and the acid concentration, and expressed as uEq/100 g body w e ig h t/h r .

4.2.3 Materials and data analysis

Atropine sulfate, verapamil HC1, bethanechol, and dopamine HC1 were purchased from Sigma Chemical Company (St. Louis, Missouri), cysteamine from Aldrich Chemical Company (Milwaukee, Wisconsin), and

Maalox (Rorer Pharmaceutical Corporation, Fort Washington,

Pennsylvania) from a local pharmacy. Pr-MDI was custom synthesized at

Chem Biocliem Research Inc. (Salt Lake City, Utah). All drug solutions were freshly prepared before use in saline, except for cysteamine which was dissolved in distilled water. The data are presented as the meaniS.E.M. The chi-square test was used for comparison of ulcer incidences and for perforation frequencies, and a one-way analysis of variance followed by the unpaired Student t test (Sinclair, 1988) was used for statistical analysis of ulcer severity data and gastric acid 119 secretion. The level of significance was set at P<0.05.

4.3 Results

C ysteam ine

A sin gle s.c. injection of cysteamine (425 mg/kg) produced duodenal ulcers within 24 hours in 80% of rats (12/15 rats; Table 5). In the 12 rats developing ulcers, the average number of ulcers per duodenum was 1,6±0.3, with an average ulcer size of 8.1±2.7 mm2 , and perforation occuring in 25% of these animals. The average ulcer severity score was 1.67±0.27 on a scale of 0-3. In addition to duodenal ulcers, cysteamine also produced gastric ulcers in 80% of rats, with a severity in d ex of 1.2+0.2 (Table 5). Ulcerations situated in the glandular segment were manifest as elongated hemorrhagic streaks along the ridges of the mucosal folds, while lesions in the antrum were characterized by sharply demarcated round-shaped necroses.

The single dose of cysteamine administered (425 mg/kg,s.c.) did not alter the pH of gastric fluid nor the acid concentration (Table 6).

However, since gastric fluid volume was significantly decreased by 52%, the calculated total acid output was similarly decreased by 54%. Since cysteamine did not increase gastric acidity and decreased total acid output in the present study, the antisecretory effects of the drugs studied (atropine, verapamil, and Maalox) had to be assessed against bethanechol-induced gastric acid secretion (Table 7). 120

Table 5. Effects of drug pre-treatments on cysteamine—Induced duodenal and gastric ulcers In rats.

Duodenal Ulcer Gastric Ulcer

Perforation in Treatments Incidencei Severity Ulcerated Rats Incidence Severity

Saline 12/15 1 .67±0,27 3/12 12/15 1.2010.20 ( i . p . )

Atropine 2/8* 0.25±0.16** 0/2 3/8* 0.5610.29 lOmg/kg ( s . c . )

Dopamine 3/6 1 ,0B±0.52 1/3 5/6 1.3310.28 30mg/kg ( i . p . )

Pr-MDI 6/8 1.2510.30 0/6 8/8 1.5010.14 lOmg/kg ( i . p . )

Pr-MDI 7/7 2.0010.22 1/7 5/7 1.2110.38 30mg/kg ( i . p . )

Verapamil 5/6 2.0010.47 2/5 6/6 1.3310.17 7mg/kg ( i . p . )

Maalox 5/8 0.94+0.27 0/5 6/8 1.3710.31 lmL (p .o .)

Maalox 5/8 0.9410.32 0/5 6/8 1.3110.35 2mL ( p .o .)

Maalox# 0/7** 0*** 0/7 1/7** 0.2110.21** 2mL (p .o .)

*P<0.05, **P<0.01, ***P<0.001 as compared to the sa lin e control group using unpaired Student's t test and one-way analysis of variance for for the severity square test for the ulcer incidence and perforation. #Rats were treated with 2ml Maalox antacid at 8:00, 10:00, 12:00, 14:00, and 20:00 hr. 121

Table 6. Effect of Cysteamine(425mg/kg) on Gastric Acid Secretion for 4 hrs In pyloric—ligated rats.

Volume Titratable Acid Total Output Treatments (ml/lOOg/hr) pH (mEq/L) (uEq/lOOg/hr)

Saline 0.81±0.13 1.7710.08 78.0917.51 66.20114.30

Cysteamine 0.3910.05* 1.7310.03 75.9113.75 30.4715.75* (425mg/kg)

*P<0.05 as compared to the saline control group (N=6). Table 7. Effects of drugs on bethanechol(3.2 ng/kg)-induced gastric acid secretion for 2 hours In pyloric-ligated ra ts.

No. of Acid Volume Acidity Total Output Treatment Rats (ml/100g/hr) pH (mEq/mL) (uEq/lOOg/hr)

Saline 7 2.14±0.04 1.6910.06 80.0715.69 170.85111.19

Atropine 5 0.1810.03*** 4.5210.95*** 8.0613.35*** 1.2110.59*** lOmg/kg (s.c .)

Verapamil 7 1.5410.16** 2.4410.30* 39.8813.48*** 61.8617.77*** 7ng/kg (i.p.)

Haalox 6 2.2010.09 2.5810.13*** 33.4614.31*** 74.49112.18*** 1ml (p .o .)

Haalox 7 1.9210.13 4.9110.22*** 7.6411.53*** 14.8413.36*** 2nl (p .o .)

*P<0.05, **P<0.01, ***P<0.001 as compared to sallne/bethanechol—treated group using unpaired Student's t test and one-way analysis of variance. 123

A tropine

Atropine (10 mg/kg, s.c.) significantly reduced the incidence of both duodenal and gastric ulcers Induced by cysteamine (Table 5), such ulcers occuring in 25% and 38% of rats, respectively, as compared to the

80% incidence of either type of ulcer in unproctected animals. This represents a 69% and 52% inhibition of duodenal and gastric ulcers, respectively. Atropine also reduced the severity of both duodenal and gastric ulcers by 85% and 53%, respectively, although the difference attained statistical significance only for duodenal ulcers (Table 5).

There were no dudoenal ulcer perforations in rats protected with atropine. Atropine significantly reduced the betlianechol-evoked titratable acidity by 90%, and significantly raised the pH of the stomach fluid by 63% (Table 7). Both gastric fluid volume and total acid output under the Influence of bethanechol were significantly reduced by atropine by 92% and 99%, respectively.

Propyl-methylenedloxyindene

Pr-MDI did not significantly alter the Incidence or the severity of cysteamine-induced duodenal and gastric ulcers at both doses tested (10 and 30 mg/kg, i.p.). The higher of the two doses of pr-MDI was previously shown to exert only a modest 12% inhibition of the bethanechol-induced titratable acidity and a 10% inhibition of the bethanechol-induced lowering of gastric pH (see ChapterIII). 124

Verapamil

Verapamil (7 mg/kg, i.p.) was ineffective In protecting against cysteamine-induced duodenal and gastric ulcers (Table 5), despite the significant 50% reduction in bethanechol-evoked titratable acidity and

31% elevation in pH (Table 7). Both the volume of gastric fluid and the total acid output evoked by bethanechol were significantly reduced by verapamil by 28% and G4%, respectively (Table 7).

Dopamine

Dopamine (30 mg/kg, i.p.) resulted in a 37% inhibition of cysteamine-induced duodenal ulceration (P>0.05; Table 5). Previously- published results indicate that this dose of dopamine has minimal effect on cysteamine-induced gastric acid secretion (Takeuchl et al., 1987).

Maalox

When administered 0, 6, and 12 hours following cysteamine, Maalox

(1 and 2 ml, p.o.) slightly reduced the incidence of cysteamine-induced duodenal and gastric ulcers by 21% and 6%, respectively (P>0.05; Table

5). On the other hand, when Maalox (2 ml) was administered 0, 2, 4,

6, and 12 hours following cysteamine, the ulcerogenic action of cysteamine on the duodenum was completely abolished and that on the stomach reduced by 82% (P<0.05; Table 5). Likewise, the severity of the cysteamine-induced gastric ulcers was significantly reduced by 82%

(Table 5). While a single oral dose of 1 ml Maalox significantly reduced by 58% the titratable acid concentration evoked by bethanechol and 125 elevated the pH by 34% (P<0,001), the corresponding values for 2 ml

Maalox were a 90% reduction in titratable acid concentration (P<0.001) and a 65% elevation of the pH (P<0.001) (Table 7). Total acid output evoked by bethanechol was siginficantly reduced by 56% and 91% by

Maalox 1 and 2 ml, respectively, with little change in the volume of gastric fluid (Table 7).

4.4 Discussion

Since pr-MDI exhibited significant protective effects against stress-induced gastric ulcers in rats at doses below those which affect the cardiovascular system (see Chapter III), it was only natural to extend these findings by exploring the potential protective effects of this intracellular calcium antagonist on duodenal ulcer development. The results of the present study, however, were disappointing in that pr-

MDI failed to exert a protective effect against cysteamine-induced duodenal ulcers. Subsequent experiments, therefore, were aimed at elucidating the reason for the failure of pr-MDI to protect against these ulcers. We were confident that the cysteamine model was working, since atropine (used as a positive control) significantly reduced the duodenal ulcer incidence by 69% and ulcer severity by 85% (Table 5).

The pathogenesis of cysteamine-induced duodenal ulcers include enhanced gastric acid secretion (Ishii et al., 1976; Szabo et al., 1977;

1979), Increased duodenal motility (Mangla et al., 1989; Takeuchl et al., 1987), delayed gastric emptying (Lichtenberger et al., 1977;

Tanaka et al., 1989), and decreased bicarbonate secretion by the 1 2 6

duodenal mucosa in response to acid (Brlden et al., 1985; Ohe et al.,

1988). We embarked on a systematic examination of these mechanisms, with the aim of elucidating the reason behind the failure of pr-MDI to protect against cysteamine-induced duodenal ulceration.

Several authors have demonstrated hyperacidity and duodenal ulceration in response to cysteamine (IshH et al., 1976; Szabo et al.,

1977; 1979) which could be blocked by vagotomy, atropine, or hexamethonium (Szabo et al., 1977; 1979; Takeuchl et al., 1987), and by pyloric ligation (Robert et al., 1974), thus implicating hyperacidity in the pathogenesis of cysteamine-induced duodenal ulcers. Pr-MDI (30 m g /k g , i.p.) was previously shown to exert only a modest 12% inhibition of the bethanechol-induced increase in acid concentration and a 10% inhibition of the bethanechol-induced lowering of the gastric pH

(see Chapter III). Thus, the failure of pr-MDI to protect against cysteamine-induced duodenal ulceration may be due to the weak antlsecretory action of this calcium antagonist on the parietal cell.

There are several arguments, however, against this conclusion. First, our present findings In pylorus ligated rats indicate that cysteamine reduced gastric fluid volume and total acid output without affecting acid concentration (Table 6), and these findings are in agreemnt with those of Robert et al. (1974). Second, verapamil was ineffective in protecting against cysteamine-induced duodenal ulceration (Table 5) despite its ability to significantly reduce by 50% the bethanechol-evoked increase in acid concentration and raise the pH by 31% (Table 7). Third, hypersecretion of acid induced by pentagastrin failed to produce 1 2 7 duodenal ulcers (Kirkegaard et al., 1980). Fourth, high doses of clmetldine, which significantly inhibited gastric acid secretion (Man et al., 1984; Stiel et al., 1983) and maintained intraduodenal pH at 7 following cysteamine administration (Ohe et al., 1983), did not prevent cysteamine-induced duodenal ulcer formation (Man et al., 1984; Stlel et al., 1983).

Nevertheless, the fact that cysteamine did not increase the acid concentration of the gastric contents in the present study (Table G) does not preclude a role for acid in the pathogenesis of duodenal ulcers produced by this agent, since cysteamine also reduces duodenal bicarbonate secretion in response to acid (Briden et al., 1985; Ohe et al., 1988) and thereby reduces the duodenal mucosal resistance to erosion by normal (or even subnormal) acid concentration (Ohe et al.,

1983). However, the observations discussed above when viewed collectively would suggest that, in addition to gastric acid, other pathogenic factors such as decreased duodenal mucosal bicarbonate secretion (Briden et al., 1985; OhB et al., 1988), increased duodenal motility (Mangla et al., 1989; Takeuchl et al., 1987), or delayed gastric emptying (Lichtenberger et al., 1977; Tanaka ey al., 1989) are also involved in cysteamine-induced duodenal ulceration.

Duodenal motility increases following cysteamine administration, resulting in localization of duodenal ulcers due to interference with the backflow of pancreatic and biliary alkaline secretions from the distal to the proximal duodenum (Mangla et al., 1989). Takeuchl et al. (1987) demonstrated no inhibition by dopamine (30 mg/kg',s.c.) of cysteamine- induced duodenal ulcer incidence, although ulcer severity was significantly reduced along with a decrease in duodenal hypermotillty, but without a reduction in acid secretion. This was interpreted as evidence that duodenal hypermotility contributes to the mechanism of cysteamine-induced ulcerogenesis. In the present study, the same dose of dopamine resulted in a 37% inhibition of cysteamine-induced duodenal ulcer incidence (P>0.05; Table 5), and did not reduce the ulcer severity. The discrepancy between our results and those of Takeuchl et al. (1987) regarding the effect of dopamine on ulcer severity may be due to the use of lower dose of cysteamine (100 mg/kg, s.c.) b y

Takeuchl et al. (1987). Since pr-MDI inhibits the in-vitro contractile response of the smooth muscle of the ileum to histamine and acetylcholine (Rahwan et al., 1977), it would have been expected that this drug would also inhibit cysteamine-induced duodenal hypermotility.

If such an effect did occur, its antiulcerogenic potential could have been overshadowed by the inhibition of gastric motility (see below) produced by pr-MDI in-vivo.

Since one of the pathogenetic factors Involved in duodenal ulcer formation by cysteamine is delayed gastric emptying (Llchtenberger et al., 1977; Tanaka et al., 1989), and since pr-MDI (10-30 mg/kg, i.p.) has been shown to significantly delay gastric emptying in-vivo in rats

(see Chapter III), it is entirely possible that this gastric effect of pr-

MDI may have overshadowed any beneficial inhibition of duodenal motility, thereby accounting for the failure of pr-MDI to protect against 1 2 9

cysteamine-induced duodenal ulceration. This may be further

compounded by the only modest inhibitory effect of pr-MDI on acid

secretion (see Chapter III). It is possible that cysteamine and pr-MDI act synergisticaliy to enhance gastric stasis. In the absence of adequate

inhibition of acid secretion by pr-MDI, the gastric distention resulting from the delayed gastric emptying may mechanically stimulate gastric acid and gastrin secretion (Llchtenberger et al., 1977). Alternatively, the accumulating gastric contents (regardless of increased or unchanged acid concentration) may leak gradually into the duodenum despite the delayed emptying, and cause erosion of the duodenum whose mucosal resistance has been reduced (Tanaka et al., 1989) by the reduction of bicarbonate secretion inflicted by cysteamine (Briden et al., 1985; Ohe e t al., 1988). It should be pointed out, however, that rapid gastric emptying, leading to increased duodenal acid load, may also overcome duodenal acid-neutralizing mechanisms and results in duodenal ulceration

(Lam e t al., 1982; Malagelada et al., 1980).

Most of the published findings support the conclusion that cysteamine Impairs the defense mechanism of the duodenal mucosa by inhibiting its function to neutralize acid, and by decreasing Its resistance to even small amounts of acid coming from the stomach

(Briden et al,, 1985; Ohe et al., 1988; Ohe et al., 1983). This Is supported by our finding (Table 7) that reduction of acid concentration

(and possibly also total acid output) by 90% or more was essential for inhibition of cysteamine-induced ulcerogenesis. This was achieved only with atropine and the larger dose of Maalox (2 ml) (Table 7), both of 130

which also inhibited cysteamine-induced ulcerogenesis (Table 5). It is

relevant that the antiulcer action of Maalox (2 ml) was demonstrable

only when the neutralizing properties of this antacid were continuously

available in the duodenum (0, 2, 4, 6, and 12 hours after cysteamine

administration) during the critical time of cysteamine-induced duodenal

mucosal damage which can be detected microscopically in rats as early

as 30-60 minutes after cysteamine and more definitively within 6 hours

(Pfeiffer et al., 1987a,b). Since the neutralizing action of antacids In

the non-fasted state lasts up to 3 hours (Fordtran, 1973), th e le ss

frequent administration of Maalox (0, 6, and 12 hours after cysteamine)

would not be expected to provide protection against cysteamine-induced

duodenal ulcerogenesis (Table 5). Likewise, pr-MDI, which inhibits the

bethanechol-induced increase in acid concentration by only 12% (see

C hapter III), would not be expected to restore the balance between acid

presentation to, and neutralizing capacity of, the duodenum particularly in the face of reduced duodenal mucosal defenses inflicted by

cysteamine. However, it should be mentioned that the antiulcer activity of high-dose Maalox in the present study (Table 5) may be unrelated to its antacid effect (Table 7), and may be due to stimulation of mucus or prostaglandin (Halter, 1988) secretion, affording a cytoprotective action. Thus, the fact that pr-MDI produces only a slight reduction in acid concentration (see Chapter III) may not be the reason for its failure to prevent the development of cysteamine-induced ulcers (Table

5). This reasoning is supported by the failure of verapamil and the lower dose of Maalox to protect against such ulcers (Table 5) despite 1 3 1 their ability to significantly reduce acid concentration (Table 7).

Finally, In addition to duodenal ulceration, cysteamine administration also produced gastric ulceration. Gastric and duodenal ulceration induced by cysteamine probably share at least some common

pathogenetic mechanisms, since both were inhibited by ntroplne and by

Maalox (Table 5) but not by dopamine or the calcium antagonists. On the other hand, the pathogenesis of cysteamine-induced and stress- induced gastric ulceration must differ significantly, since only the latter was inhibited by the calcium antagonists pr-MDI (at subcardiovaseular

doses) and verapamil (at supracardiovascular doses) (see Chapter III). CHAPTER V

ANTIULCER ACTIVITY OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE. EFFECTS ON GASTRIC LESIONS IN RATS INDUCED BY COLD-RESTRAINT STRESS AND THYROTROPIN-RELEASING HORMONE.

5.1 Introduction

The cold-restraint stress-induced gastric ulceration model in rats has been widely used to test the antiulcer activity of various pharmacological agents (Pare and Glavin, 1986). The acute gastric superficial hemorrhagic mucosal lesions in rats highly resemble the human acute stress ulcers in patients with severe injury or Illness (Kleiman et al,, 1988; Silen, 1988). The pathogenesis of gastric lesions In this cold- restraint model is not fully understood; however, many studies have suggested that acute stress ulcer is primarily mediated by a marked increase In gastric muscular contractility (Garrick et al., 1986a,b; Ogle et al., 1985) and reduction of gastric mucosal blood flow (Kleiman et al,,

1988; Robert et al., 1989). To the contrary, gastric acid secretion, which is enhanced, unaffected, or suppressed by cold-restraint stress

(Garrick et al., 1986a; Dai and Ogle, 1974; Cho and Ogle, 1979), has been relegated a less significant role in stress gastric lesion formation

(Garrick et al., 1986a; Cho and Ogle, 1979; Cho et al., 1985).

132 1 3 3

Although calcium ion plays a pivotal role at various etiological steps

in the development of gastric ulcer (Wolfe and Soil, 1988), the

experimental and clinical results with a variety of calcium channel

blockers have been inconsistent and conflicting (Kleiman et al., 1988;

Spedding, 1988). This is possibly due to the uncertainty concerning the

existence or type of membrane calcium channels In parietal cells

(Spedding, 1988; Wolfe and Soil, 1988), and the lower sensitivity of

calcium channels in gastrointestinal smooth muscle to calcium channel

blockers as compared to cardiovascular calcium channels (Spedding,

1988). In an attempt to circumvent the complexities and uncertainties of

the gastrointestinal membrane calcium channels, we examined the potential antiulcer activity of propyl-metliylenedioxylndene (pr-MDI), an intracellular-acting calcium antagonist (Rahwan, 1989), which inhibits calcium mobilization from the endoplasmic reticulum (Wong and Rahwan,

1988). Our findings demonstrated the ability of pr-MDI to prevent the development of cold-restraint stress-induced gastric ulcers in rats in a dose-dependent manner at doses (10-30 mg/kg, I.p.) significantly lower than those required to exhibit antinrrhythmlc activity. The inhibitory effect of pr-MDI on the development of gastric stress ulcers was highly correlated with its inhibitory effect on gastric motor activity (see

C hapter III).

Recently, neuropharmacologlcal studies have demonstrated that intracisternal or intracerebroventricular microtnjection of thyrotropin- releasing hormone (TRH) rapidly and markedly increased gastric acid secretion, gastric contractility and emptying, and gastric mucosal blood 1 3 4 flow, and promoted gastric ulcer formation (Tahce et al., 1989). The central action of TRH on gastrointestinal function regulation was mediated by increased efferent parasympathetic outflow from the dorsal vagal complex (Tache et al., 1989a,b). It has been shown that TRH was released in the median eminence of rats upon exposure to cold (Aranclbia e t al., 1983), and the TRH was proposed to be the brain mediator responsible for cold-restraint stress gastric ulcers in rats (Tache et al.,

1989a, b). The purpose of the present study was to evaluate the inhibitory actions of pr-MDI on TRH-induced increases In gastric acid secretion, gastric emptying, and gastric ulceration, and to correlate the antiulcer activity of pr-MDI in the cold-restraint stress model and the

TRH-induced gastric lesion model.

5.2 Methods and Materials

Male Sprague-Dawley albino rats, weighing 210-360 g, were maintained on Purina Laboratory Chow and tap water ad libitum, and housed in facilities at controlled temperature (70±2)°F with 12-hr light- dark cycle. Prior to all experiments, the rats were fasted for, 24 hrs with free access to top water, while being housed in individual cages with raised wire mesh bottom (to prevent coprophagy).

5.2.1 Cold-restraint stress ulcers

The fasted rats were injected intraperltoneally (i.p.) with either saline or pr-MDI (30 mg/kg) 10 mins before they were Immobilized in individual restrainers and exposed to cold (4°C) for 3 hrs In a dark 1 3 5 ventilated cold room. Within 10 mins after the cold/restraint stress the rats were sacrificed with excessive CO 2 inhalation, and the stomach quickly excised, washed, and opened along the greater curvature. The open stomachs were rinsed under tap water, and the gastric mucosa examined for ulcerations with the aid of a stereomicroscope (lOx). The number of ulcers and the lesion length (L) and width (W) were recorded. The results were expressed as ulcer incidence, average number of ulcers per stomach, and average ulcer size (mm2) which is calculated as an ellipse (L x W x t i/ 4 ) .

5.2.2 TRH-lnduced gastric ulceration

The fasted rats were treated i.p. with either saline or pr-MDI (30 mg/kg) 10 mins before they were lightly anesthetized with ethyl ether and injected Intracisternally (i.e.) with the stable TRH analogue,

RX77368 (100 ng), via a Hamilton microliter syringe (Hamilton Co.,

Reno, Nevada). The i.e. injections were performed with a stereotaxic apparatus. The rats were then returned to their individual cages with no food or water for 3 hrs (Nakane et al., 1985). The conscious animals were then sacrificed with ether overdose and the stomach quickly isolated, washed, and opened along the greater curvature. The lesion measurements were the same as described above.

5.2.3 TRH-induced gastric emptying

Pasted rats were pre-treated i.p. with either saline or pr-MDI (30 mg/kg) 10 mins before thei.e. administration of RX77368 (100 ng), while 136

under light anesthesia. As soon as the animals recovered from

anesthesia, they were orally fed a 1.5 ml liquid test meal containing

methylcellulose and phenol red dye (Walsh, 1989). Twenty mins after

the meal, the animals were sacrificed by ether overdose. A separate

group of rats (controls) was killed Immediately after the administration of

the test meal, and the data obtained from this group (representing 0%

gastric emptying) was used for calculations. After sacrifice and

laparotomy, both the pylorus and the cardia of each stomach were

ligated, and each stomach was removed and homogenized in 10 ml 0.1 N

NaOH solution. The homogenates were centrifuged at 5,000 xg for 10

minutes at 4°C. To each supernatant was added 1 ml of 20%

trichloroacetic acid to precipitate the proteins, followed by centrifugation

at 1,500 xg for 15 minutes. The supernatant from the centrifugation was

then mixed with 2 ml of IN NaOH to maximize the color intensity. An

aliquot (1 ml) of the sample was diluted in 2 ml of distilled water, and

the analysis of phenol red retention was the same as previously

described (see Chapter HI).

5.2.4 TRH-induced gastric acid secretion

Pyloric-ligatlon was performed as described in Chapter III.

Briefly, the fasted rats were treated i.p. with either saline or pr-MDI

(30 mg/kg) 10 mins before thei.e. injection of E.X77368 (100 ng), while under light anesthesia. Immediately after UX77368 administration, the pylorus was ligated through a small abdominal incision. The abdomen was then closed and the ether discontinued. The rats were then 137 returned to their cages with no food or water for 2 hrs (Tache et al.,

1985), and were subsequently killed with ether overdose. The stomach was removed after an additional ligature was placed around the esophago-cardiac junction. A puncture was made in the rumenal segment along the greater curvature, and gastric contents were collected, centrifuged, and analyzed for acid content as previously described (see

C hapter III).

5.2.5 Materials and data analysis

RX773G8 (Reckitt and Colman, Kingston upon Hill, U.K.) in pow der form was dissolved in 0.9% saline and kept at -20°C. Before each experiment, the peptide was diluted with saline and Injected i.e. In a volume of 10 ul into rats under ether anesthesia. Methylcellulose (400 centipoises) was purchased from Sigma Chemical Co.(St. L ouis,

Missouri), and phenol red from Coleman and Bell Co. (Norwood, Ohio).

Pr-MDI was custom synthesized at Chem Blochem Research Inc. (S alt

Lake City, Utah) and the drug solution was freshly prepared in saline before use, and administered in a volume of 2 ml/kg body weight.

The data are presented as the mean+ S.E.M.. The Chi-square test was used for comparison of ulcer incidence, and a one-way analysis of variance followed by the unpaired Student's t test (Sinclair, 1988) was used for statistical analysis of number of ulcers and ulcer area, gastric emptying, and gastric acid secretion. The level of significance was set at

P<0.05. 1 3 8

5.3 Results

5.3.1 Cold-restraint stress ulcers

The ulcers arising from the cold-restralnt stress were exculsively localized in the glandular portion of the stomachs of the rats. They were manifested as black focal hemorrhagic erosions and elongated hemorrhagic streaks along the ridges of the mucosal folds. Pre-treatment of rats with pr-MDI (30 mg/kg) significantly reduced the number of ulcers per stomach and the ulcer area by 69% and 86%, respectively, without affecting the ulcer incidence as compared to the saline-treated group

(T able 8 ).

5.3.2 TRH-induced gastric ulcerations

Intraclsternal injection of the vehicle into the rats did not result in gastric ulceration while a single I.e. injection of RX77368 (100 ng) produced gastric ulcers within 3 hrs in 75% of the treated rats with an average of 4.4410.73 ulcers per stomach and an average ulcer area of

1.1910.30 mm2 (Table 9). RX77368-induced gastric ulcers were less severe than those produced by cold-restralnt stress, and the ulcers were mainly localized in the glandular portion of the stomach. Pre- treatment of rats with pr-MDI (30 mg/kg) did not significantly reduce the number of ulcers or the ulcer area induced by RX77368, while the ulcer incidence was increased to 92% (P>0.05) (Table 9). The

RX77368-induced gastric ulcers in pr-MDI-treated rats were generally localized at the distal portion of the glandular stomach. Table 8. Effect of pr-MDI on cold-restralnt stress-induced gastric ulcers In rats.

No. o f U lcer No. of Ulcers U lcer Area Treatments Rats Incidence per Rat (mm sq.)

S a lin e 5 5 /5 14.8012.08 8.59±2.97

Pr-MDI 5 5/5 4.6011.47** 1.2210.59* (30 mg/kg)

*P<0.05, **P<0.01 as compared to the saline-treated group. 140

Table 9. Effect of pr-MDI on gastric erosions In rats Induced by Intraclstem al administration of the TRH analogue, RX77368.

Pre- No. o f U lcer No. o f U lcer Area Treatments Ulcerogen Rats Incidence U lcers/R at (mm sq.)

Saline Vehicle 4 0/4 0 0

S a lin e RX77368 12 9/12 4.4410.73 1.1910.30 (lOOng)

Pr-MDI RX7736B 12 11/12 3.1810.52 0 .8 1 1 0 .2 2 (30 mg/kg) (lOOng) 1 4 1

5.3.3 TRH-induced gastric emptying

The effects of pr-MDI on basal and RX77368-induced gastric

motility, measured as gastric emptying, are shown in Table 10. Gastric

emptying recorded 20 mins after the methylcellulose meal in saline-treated

rats was 58%. Pre-treatment of rats with pr-MDI (30 mg/kg) delayed the

basal gastric emptying to 27.95%; however, the inhibition (=52%) was not

statistically significant. On the other hand, a single i.e. injection of

RX77368 (100 ng) significantly accelerated the gastric emptying of the

test meal to 96.12% in 20 mins, which is a 66% increase as compared to

the saline-treated group. Pr-MDI (30 mg/kg) significantly blocked the

enhanced gastric emptying induced by RX77368 to the level of basal

gastric emptying.

5.3.4 TRH-induced gastric acid secretion

As compared to the unstimulated rats, a single i.e. injection of

RX77368 significantly increased gastric secretion volume (P<0.001), titratable acidity (P<0.001) and total acid output (P<0.001), and reduced the pH of the gastric secretion (P<0.01) (Table 11). Pr-MDI (30 mg/kg) abolished the effect of RX77368 on all of these parameters without influencing basal levels.

5.4 Discussion

Acute gastric ulcerations developed in the glandular portion of the stomach in all rats exposed to cold-restraint stress. Pr-MDI (30 mg/kg) significantly reduced the number of cold-restralnt stress ulcers per rat Table 10. Effect of pr-MDI on gastric emptying in rats treated in t r a c is te m a lly with the TRH analogue, RX77368.

Pre- No. o f Treatments Ulcerogen Rats G astric Emptying (X)

S alin e Vehicle 6 58.00±8.30

Pr-MDI(30 rag/kg) V ehicle a 27.95±10.77

S alin e RX77368(lOOng) 6 96.1210.29//

Pr-MDI(30 mg/kg) RX77368(lOOng) 8 59.74110.55*

//P<0.05 as compared to s a lin e /v e h ic le control group. *P<0.05 as compared to saline/RX77368 treated group. 1 4 3

Table 11. Effect of pr-MDI on gastric acid secretion in pyloric-ligated rats treated in tr a c is to m a lly with the TRH analogue, RX77368.

Gastric Vol. A cidity Total Output Treatments N (ml/lOOg/hr) pH (mEq/10 (uEq/lOOg/hr)

Saline & Saline 5 0.4410.02 2.6610.21 33.9214.07 15.2512.14

Pr-MDI(30mg/kg) 4 0.3610.06 2.7110.22 39.8014.21 14.0712.70 & S alin e

Saline 6 1.3310.08//// 1.4010.04// 92.1412.59//// 122.1413.64//// & RX77368(lOOng)

Pr-MDI(30mg/kg) 6 0.4110.05** 2.8510.40> 33.3517.73** 15.3015.92** & RX77368ClOOng)

//P<0.01, ////P<0.001 as compared to the s a lin e /s a lln e control group. *P<0.01, **P<0.001 as compared to the saline/RX77368 treated group. 1 4 4 without influencing the ulcer incidence (Table 8). These results confirm our previous findings (see Chapter III). Many studies suggested that luminal hydrochloric acid does not play a major role in cold-restraint stress ulcer formation, since total neutralization of luminal acid did not prevent ulcer development induced by cold-restraint stress (Dai and

Ogle, 1974; Cho and Ogle, 1979; Cho et al., 1985), and, in many instances, gastric acid secretion was significantly suppressed by cold- restraint treatment (Garrick et al., 1986a; Dai and Ogle, 1974; Cho and

Ogle, 1979). Other findings indicated that high amplitude contractions of long duration developed in the stomach are the major contributing factor in the etiology of stress ulcers in rats (Garrick et al., 1986a;

Ogle et al., 1985; Cho et al., 1985; Koo et al., 1989). The enhanced gastric muscular contractility may result in mechanical restriction of gastric mucosal blood flow, leading to focal ischemia and ulceration

(Garrick et al,, 1986a; Cho et al., 1985), Garrick et al. (1986a,b) demonstrated that papaverine HCI suppressed the cold restraint-induced gastric contractility and the subsequent gastric lesions without affecting gastric acid secretion. Pr-MDI has been shown to exhibit its protection against cold-restraint stress ulcers by inhibiting gastric motility (as measured by the delay in gastric emptying) In a dose dependent manner

(10-30 mg/kg; see Chapter III). Moreover, gastric contractility stimulated by cold-restralnt stress was shown by direct measurement to be inhibited by pr-MDI (30 mg/kg) by >90% (personal communication from

T. Garrick). The inhibitory action of pr-MDI on gastric muscular contractility Is probably associated with its intracellular calcium 145

antagonistic action on gastric smooth muscle since cold-restraint stress-

induced gastric contractility is regulated by muscarinic- and Hi-receptor

activations which, in turn, mediate increases in intracellular calcium

concentration (Ogle et al,, 1985; Cho and Ogle, 1979; Koo et al., 1989).

These findings suggest that pr-MDI exerts its antiuleer activity mainly

through inhibition of gastric contractility, and supports the contention

that increased gastric contractility plays a major role in ulcerogenesis in

the cold-restraint stress model (Garrick et al., 1 9 8 6 a ,b ).

Growing evidence has demonstrated that central administration of

the neuropeptide TRH in rats significantly increases gastric acid

secretion, gastric emptying and contractility, and gastric mucosal blood

flow, and promotes gastric ulcer formation (Tache et al., 1 9 8 9 a ,b ).

These stimulatory actions of TRH are centrally mediated through vagal-

dependent cholinergic pathway, which can be blocked by vagotomy or

atropine (Maeda-Hagiwara et al., 1987; Garrick et al., 1987; Yoto and

Tache, 1985; Theifin et al., 1989). Many studies support a role of

endogenous central TRH in the ulcerogenesis of rats exposed to cold-

restraint stress (Tache et al., 1989a,b). The present study, however,

revealed that pr-MDI (30 mg/kg) did not significantly reduce the

RX77368-induced gastric ulceration in rats (Table 9), but It almost

abolished the RX77368-induced acceleration of gastric emptying of a

methylcellulose meal (Table 10), as well as the RX77368-lnduced Increases

in gastric acidity and acid output (Table 11). Moreover, pr-MDI (30

mg/kg) inhibited the RX77368-induced increase in gastric contractility by

>75% (personal communication from T. Garrick). The antisecretory and anti-gastric motility actions of pr-MDI in this model are likely mediated hy its intracellular calcium antagonistic effect at both central and peripheral levels. Since TRH receptor activation in pituitary cells

Involves intracellular calcium mobilization from the endoplasmic reticulum

(Mollard et al., 1989), which is the identified target for the Intracellular inhibitory action of pr-MDI (Wong and Rahwan, 1988), it would be expected that pr-MDI would block the TRH-receptor mediated calcium mobilization in cells of the dorsal vagal complex, resulting in decreased vagal efferent discharges and subsequent gastrointestinal activities.

Alternatively, pr-MDI might block the release of acetylcholine from the cholinergic nerve terminals since It is a calcium dependent process (Wolfe and Soil, 1988). In addition, pr-MDI might also participate in inhibition of excitation-contraction coupling of gastric smooth mucsle (Rahwan,

1989; Koo et al., 1989). Although it has been demonstrated that TRH- induced gastric ulceration was blocked by antisecretory doses of cimetidine and omeprazole (Goto and Tache, 1985; Larrson et al., 1983), other unidentified ulcerogenic factors must be involved since neither bethanechol nor histamine, given at equivalent secretory doses, produced gastric ulcerations (Tache et al., 1989b; Maeda-Hagiwara et al., 1983).

Moreover, It has been suggested that inhibition of acid secretion by more than 90% is required to prevent gastric ulcerations in the cold-restralnt stress model (Garrick et al., 1987). The present findings demonstrate that blockade of TRH-induced gastric contractility does not prevent gastric ulcer formation by this peptide, and therefore, other ulcerogenic mechanisms might play a major role In TRH-induced gastric lesion 147

formation. Moreover, substantial reduction of gastric acidity (>90%) might

be required for effective antiulcer activity in this model system.

In summary, cold-restraint stress and central injection of TRH

invariably produced gastric lesions in rats within 3 hrs. Although the

ulcerogenic mechanism of these two models is mainly mediated by

enhanced cholinergic activation, the major contributing factor for ulcer

formation may vary. Cold-restraint stress increases gastric contractility

and decreases mucosal blood flow, leading to focal Ischemia and ulcer

formation (Garrick et al., 1986a,b; Ogle et al., 1985; Robert et al.,

1989). On the other hand, central injection of TRH increases, rather

than decreases, gastric mucosal blood flow, which excludes the

possibility of ulcer formation due to Ischemia of gastric mucosa (Tache et

al., 1989a). The present findings suggest that enhanced gastric

contractility may play a major role in cold-restraint stress-induced

gastric ulcer formation, while sustained gastric acid secretion, together

with other unidentified factors, may contribute to the TRH-induced

gastric ulceration. Furthermore, these findings suggest a possible

dissociation between the ulcerogenic mechanisms of cold-restraint stress and intracisternal administration of TRH. r

CHAPTER VI

SUMMARY AND CONCLUSIONS

Propyl-methylenedioxyindene (pr-MDI) is an intracellularly-actlng calcium anatagonist with cardiac H2-receptor blocking properties.

Excitation-contraction coupling of the gastrointestinal smooth muscle in vitro induced by histamine (Hi-receptor mediated) and acetylcholine

(muscarinic receptor mediated) is blocked by pr-MDI. Stimulus- secretion coupling in inhibited by much lower concentrations of pr-MDI than is excitation-contraction coupling. Since the processes leading to gastric ulceration are calcium-dependent, we examined the potential antiulcer activity of pr-MDI in various experimental ulcer models In ra ts.

The first phase of the present Investigation was to examine the antiulcer activity of pr-MDI (10, 20, and 30 mg/kg) and to it compare with that of cimetidine (10, 20, and 30 mg/kg) and verapamil (11, 16, and 32 mg/kg) in the cold-restralnt stress-induced ulcer model in male rats. The rats were deprived of food for 24 hours prior to restraining and exposure to cold (4°) for 3 hours. Drugs were administered intraperitoneally 10 minutes prior to the cold-restraint stress. The mean number of stomach ulcers per rat (in round figures) was reduced from

15 (vehicle control) to 10, 5, and 2 (pr-MDI), 3, 4, and 1 (cimetidine),

1 4 8 and 8, 5, and 1 (verapamil), in the ascending order of their doses.

The mean cumulative length (mm) of ulcerated stomach surface was

reduced from 16 (vehicle control) to 9, 6, and 2 (pr-MDI), 2, 2, and 1

(cimetidine), and 7, 4, and 1 (verapamil), in the ascending order of

their doses. The highest antiulcer dose of pr-MDI (30 mg/kg) was lower

than its antiarrhythmic dose in rodents (antiarrhythmic EDso: 53-84

mg/kg). Since the intraperitonoal LDso values in rats for pr-MDI and

verapamil are 175 mg/kg and G7 mg/kg, respectively, the equivalent

antiulcer activity of these drugs was achieved at one-ninth of the LDso

dose of pr-MDI (20 mg/kg) and one-fourth of the LD50 dose of

verapamil (16 mg/kg). At one-eighteenth of its LDso (10 mg/kg) pr-MDI

afforded significant protection against ulceration. It is concluded that

pr-MDI possesses antiulcer properties against cold-restraint stress-

induced gastric ulcers in rats at doses (10-30 mg/kg,i.p.) significantly

lower than those required to exhibit cardiovascular effects. It is also

suggested that the H2-blocking property of pr-MDI[pAi=G.46 in the Hz-

coupled cardiac adenylate cyclase assay as compared to a pAz of 6.10

for cimetidine] was not the sole or major mechanism of the antiulcer

effect. This is because the pAz value for verapamil-induced Hz blockade

under the same conditions was 5.44, while the equivalent antiulcer

doses of pr-MDI and verapamil were 20 mg/kg and 16 mg/kg, respectively. It is, of course, possible that the cardiac and gastric Hz

receptor may respond differently to these drugs.

The second phase of the present investigation was to determine the antiulcer mechanisms of pr-MDI by examining its inhibitory actions on 150

gastric acid secretion, mast cell degranulation, and gastric motility in

rats. Since cold-restraint stress ulcers is primarily mediated by

cholinergic overactivity, and since, in many instances, gastric acid

secretion is suppressed by cold-restraint stress, bethanechol-Induced

hydrochloric acid secretion in acutely pylorus-ligated rats was used as

a model to examine the antisecretory action of pr-MDI. At 30 mg/kg

(i.p.), pr-MDI significantly obtunded bethanechol-induced HC1

secretion, but the effect was significantly weaker than that produced

by verapamil (16 mg/kg, i.p.) or cimetidine (10 mg/kg, I.p.). Since

30 mg/kg pr-MDI produces greater antiulcer activity than the very high

dose of 16 mg/kg verapamil, it is unlikely that Inhibition of acid

secretion plays more than a contributory role in the antiulcer mechanism

of action of pr-MDI.

Although the present investigation unfortunately failed to verfiy

the inhibitory action of pr-MDI on bethanechol-mediated mast cell degranulation due to errors occurring during histological processing of

the gastric sample, the gastric motility study revealed significant findings In that pr-MDI (10-30 mg/gk, i.p.) produced a dose- dependent slowing of gastric emptying in rats fed a methylcellulose/phenol red test meal, and this effect correlated well with the antiulcer action as evidenced by a correlation coefficient close to unity. Moreover, pr-MDI (30 mg/kg) was shown to inhibit cold- restraint stress-induced gastric contractility by 90% using transducer measurement (personal communication from T. Garrick). Verapamil (16 mg/kg, i.p.) did not affect gastric emptying. It is concluded that 1 5 1

reduction of gastric motility plays a major role in the mechanism of the antiulcer action of pr-MDI.

The third phase of the Investigation was to explore the antiulcer action of pr-MDI on cysteamine-induced duodenal ulcers at the same low doses. Duodenal ulcers were induced in rats with a single dose of cysteamine (425 mg/kg, s.c.), which produced an 80% ulcer incidence within 24 hours without affecting gastric acid concentration. The following drugs were administered at 0, 6, and 12 hours post- cysteamine in an attempt to prevent duodenal ulcer development:

Atropine (10 mg/kg, s.c.; which Inhibits gastrointestinal motility and reduces acid concentration); pr-MDI (10 and 30 mg/kg, i.p.; which reduces gastrointestinal motility with only modest reduction in acid concentration); verapamil (7 mg/kg, i.p.; a calcim channel blocker which reduces acid concentration without affecting motility); dopamine

(30 mg/kg, i.p.; which inhibits duodenal motility with minimal effect on acidity); and Maalox (1 and 2 ml, p.o.; an antacid). Atropine reduced the cysteamine-induced duodenal ulcer incidence by 69%. None of the other drugs significantly reduced the ulcer incidence. Duodenal ulcer development was abolished when Maalox (2 ml, p.o.) was administered at 0, 2, 4, 6, 12 hours post-cysteamlne which ensured continuous presence of acid-neutralizing activity in the duodenum. Atropine and

'continuous* Maalox administration reduced gastric acid concentration by

> 90% (when acid secretion was stimulated by bethanechol). The failure of pr-MDI to protect against duodenal ulcerogenesis Is discussed in relation to four proposed pathogenic mechanisms of action of cysteamine: 152 hyperacidity, increased duodenal motility, delayed gastric emptying, and reduction o£ duodenal acid-neutralizing capacity. The delayed gastric emptying produced by pr-MDI may overshadow any decrease in duodenal motility, and this may be compounded by the inability of the drug to significantly reduce acid concentration or affect duodenal acid- neutralizing activity.

The fourth phase of the present investigation was to examine the inhibitory action of pr-MDI on TRH-induced gastric ulceration and to correlate the antiulcer activity of pr-MDI in the cold-restraint stress model and the TRH-induced gastric lesion model. Pr-MDI (30 mg/kg) did not reduce the gastric ulceration induced by intraclsternal administration of 100 ng RX77368, a stable TRH analogue, even though it abolished the RX77368-induced stimulation of gastric motility, measured as gastric emptying, as well as the RX77368-induced increases in gastric acidity and acid output. Since pr-MDI (30 mg/kg) significantly inhibited stimulation of gastric motility by both cold- restraint stress and TRH, but only cold-restraint stress ulcers were effectively reduced by the drug, the present findings suggest a possible dissociation between the ulcerogenic mechanisms of cold- restraint stress and intraclsternal administration of TRH. REFERENCES

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